Biochhnica el Biophysica Acta, 1071 (1991) 473-501 © 1991 Elsevier Science Publishers B.V. A l l rights reserved 0304-4157/91/$03,50

473

BBAREV 85392

Reconstitution of receptor/GTP-binding protein interactions Richard A. Cerione Department of Pharmacology, Schurman Hall, Corneil University, Ithaca, N Y (U.S.A.) (Received 21 January 1991}

Contents I,

Introduction

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II, $oAdronergtc ret~eplor/G~ systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, G~n~ral reconstitation approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B, Assays of ~oadrenergic receptor/O~ interactions in reconstituted phospholipid vesicle systems. I, High-affinity binding of agonists to rt~'ceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, High-affinity binding by guanine nucleotides to reconstituted vesicles containing the $-adrenergie receptor and G~ proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. Correlation of high-affinity binding of GTP~.S and the activation of the G~ protein . . . . ii. Dependence of the isoproterenol-stimulated [3sS]GTPTS binding on the concentration of GTP3,S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii. Effects of Mg 2 + on the binding of ['~sS]GTP ~,S to reconstituted ~-adrenergic receptor/G, vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv. Possible scheme describing the high-affinity binding of guanine nucleotides to G~. . . . . . v. Role of GDP dissociation in the agonist-stimulated binding of GTP to G s . . . . . . . . . . . 3, Assays of hormone-stimulated GTPase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Use of reconstitution and molecular biology approaches to map key domains involved in receptor/G protein interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Receptor studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. Molecular biology studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii. Trvptic digestion studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill. Rhodopsin/transduein system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Reconstitution studies/GTPase measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fluorescence studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Changes in aT fluorescence as a function of [rhodopsin] and [GTP] . . . . . . . . . . . . . . . . . . 2. Changes in aT fluorescence as a function of [/]:,T] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mechanistic implication~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

474 475 475 476 476 478 478

478 479 479 479 480 481 481 481 484 485 485 480 486 487 489 4~1

IV. Growth factor receptor tymsine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Insulin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Insertion of the insulin receptor tyrosine kinase into phospholipid vesicles . . . . . . . . . . . . . 2. Insulin-stimulated phosphorylation of G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. E G F receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

492 493 493 ,194 495

V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

498

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

498

Correspondence: R.A. Cerione, Dept. Pharmacology, Schurman Hall, Cornell University, Ithaca, NY 14853, U.S.A.

474 1. Introduction

it has been well documented that the regulation of a number of second messengers occurs via the coordi: nated interactions between proteins comprising receptor-coupled signal transduction systems. In many of these systems, the second messenger regulation is the outcome of interactions occurring between three protein components; specifically, a cell surface receptor protein, a GTP-binding transducer protein (G protein), and an effector protein which can be an enzyme or an ion channel. Among the types of cellular second mes~ngers that are regulated in this manner are cyclic AMP via the hormonal regulation of adenylyl cyclase [1,2], inositol trisphosphate and diacylglycerol via the receptor-activation of phospholipase C.enzymes [3-6], cyclic GMP through the light-stimulated phototransduction pathway operating in vertebrate vision [7,8], arachidonic acid through the receptor-activation of phospholipase A, [9,10], and various ions (e.g. K +, Ca'*) through the regulation of voltage-dependent ion channels [11-19]. A great deal of research effort has been invested in delineating the molecular mechanisms underlying these different receptor-coupled signaling systems. This, at least in part, is due to the expectation that a molecular understanding of these signaling cascades will be directly relevant to the development of therapeutic strategies for a number of disease states. The effective reconstitution of cell surface receptor proteins with the other members of their signal transduction pathways represents an important first step towards characterizing the molecular meclmnisms underlying these signaling systems, This is especially true since in many cases the simple mixing of a purified hormone receptor (such as the/3-adrenergic receptor) with a purified O protein (such as the O~ protein) in detergent solution will not result in the functional coupling of these proteins, i,e,, detergents markedly interfere with reccptor/G protein interactions. Thus in order to monitor the functional coupling between the purified O-adrenergic receptor and the purified G~ protein, it was necessary to develop procedures for removing the detergents from these proteins and insetting the proteins them into phospholipid vesicles. This also was necessary when assaying hormonalstimulated adenylyi cyclase activity using the purified ~-adren©rgic receptor, the G~ protein, and the adenvlyl cyclase enzyme [20], The construction of phospholipid vesicle systems containing a purified receptor protein, a G protein, and an effector enzyme in principle should provide the potential for unambiguously determining the rate-limiting steps for the receptor-stimulated activation (GTPbinding) and deactivation (GTPase) of the G protein and for the regulatory interaction of the activated G protein with the effector protein. This stems from the

fact that phospholipid vesicle systems containing purified signaling components are amenable to experiments where the amounts of the individual protein components of the system are varied in a well-defined manner. The use of reconstitution approaches also provides for the possibility of identifying key functional domains on the individual components of the signaling system. For example, the specific modification of a protein component by protease digestion, chemical modification, or site-directed mutagenesis, followed by the reconstitution of the modified component with other native (unmodified) components of the signaling system, enables the assessment of the functional importance of different amino acid side-chains or peptide domains in the protein interactions comprising the signaling event. Over the past several years, a number of receptor/G protein-coupled signaling systems have been functionally reconstituted. The first of these was the/3-adrenergic receptor/G~-coupled pathway. While the initial reconstitutions of this signaling system were performed using partially purified preparations of the /3-adrenergic receptor [21-23], in a relatively short period of time reconstitution systems were established using highly purified preparations of this receptor and the G~ protein [24-33]. Almost in parallel, similar types of reconstitution studies were being performed using highly purified components of the vertebrate vision system [29,34,35], and then subsequently, other reconstituted receptor/G protein systems were developed including systems comprised of the a2-adrenergic receptor and the G i proteins ['~6,37], the muscarinic acetylcholine receptor and the Gi and G o proteins [38-41], as well as reconstituted systems containing dopamine receptors [42] or GABA receptors [43] and different G proteins. In addition, procedures are now being developed to examine the interactions between various growth factor receptors and candidate transducers for mitogenic signaling pathways [44-46]. Overall., the reconstitutions of different receptor/G protein-coupled signaling pathways have provided important information regarding the detailed sequence of events that are responsible for the receptor-promoted binding of GTP to the G protein, the activating conformational changes that accompany GTP binding, the functional coupling of the activated G protein to the effector protein, and the mechanisms important in the regulation of G protein-deactivation (i.e., GTPase activity). In the cases of the growth factor-coupled signaling systems, these reconstitution approaches should also provide important information regarding t-he identity~ of the protein components participating in the mitogenic signaling pathways. Once the various components have been identified, it should then be possible to obtain information regarding the molecular mechanisms underlying these signaling pathways. It especially

475 will be of interest to determine whether any analogies exist between the traditional hormone receptor-mediated signaling pathways (i.e. those involved in the regulation of adenylyi cyclase or phospho!ipase C) and those operating in the regulation of cell growth. In this review, the types of mechanistic information that have been obtained for hormone receptor/G protein coupling will be considered with the primary emphasis being placed on /3-adrenergic rec,~pto~/GJ adenylyl cyclase interactions. Within this section, both steady-state kinetic information, that largely has been derived from experiments measuring the hormone/ receptor-stimulated binding of radio-labeled guanine nucleotides to G~ or the hydrolysis of 32P-labeled GTP, as well as information pertaining to the identification of key functional domains involved in /3oadrenergic receptor/G~ interactions that has come from molecular biology studies will be presented. This will be followed by a coverage of recent reconstitution studies using the purified components of the vertebrate vision system, where fluorescence spectroscopic approaches have been used to monitor the individual steps in the phototransduction signaling ca:;cade in real time. Finally, the types of reconstitution systems that are now being used to address the question of whether GTPbinding proteins functionally couple to growth factor receptor tyrosine kinases will be considered.

11. ~-Adrenergic receptor/G~ systems The/3-adrenergic receptor-coupled adenylyl cyclase system has served as a prototype for hormone recept o r / G protein-coupled signaling pathways. This system operates through the interactions of three, membranebound proteins; specifically, the/~-adrenergic receptor itself, a single chain glycoprotein of M, = 64000 [47,48], the GTP-binding protein, G~, a heterotrimer with its subunits designated as a~ (Mr---42-52000), fl (hi, = 36000), and y (Mr---5-10000) (49-51), and adenylyl cyelase (also a single chain glycoprotein of M,= 150000) [52]. The initial step in this signaling pathway involves the interactions of a hormone-bound/~-adrenerg.~e receptor with the G~ protein, which then results in the exchange of a tightly bound molecule of GDP (on the a~ subunit) for GTP. This exchange is referred to as the activation event. !t has been commonly suggested that the GTP-bound, activated a~ then dissociates from both the hormone-bound /3-adrenergic receptor complex, and from the 8~ subunit complex (which stays intact under all non-denaturing conditions), and goes on to interact with adenylyl cyclase [53-56]. The a~GTP species will persistantly stimulate cAMP production by adenylyl cyclase until the bound GTP is hydrol~,zed back to GDP. This GTPase activity serves to deactivate the a~ subunit, resulting in its

reassociation with the /3r subunit c(~mplen, ~hereb~ returning the ~stem to its starting poinlo

11-,4. General reconst#ution approaches Reconstitution approaches have been used lo examine many of the steps ~ccurring in ~he /~oadre~ergic receptor-coupled adenylyl cyda.~ signaling pathway. Specifically, two component phoxpholipid vesicle systems comprised of the purified /3-adrenergic receptor and the G, protein have been constructed and used to examine ~he receptor-stimulated actk,ation-deacfivation cycle of the G~ protein [23,24,26-30,33], whereas more complex systems, containing three-five components, have been constructed to study the fl-adrenergic stimulation of adenylyi c'yclase activity as well as the modulation of that activity by activated forms of ~he inhibito~ GTP-binding protein, G i [~,28,31,32,57-59]. The specific details of the reconstitution procedures used to assay these different receptor/G,-coupled activities have been elaborated upcn in other revie~.~ [43°60-62]. In this re,hew, detailed reconstitution protocols are not presented, but rather the rationale behind the general procedure that was dcwctoped for reconstituting both receptor/G protein and receptor/ G protein/effector interactions is described. The basic strategy of the reconstitution approach is to open or solubilize a lipid vesic|e structure and then to mix the solubilized vesicles with purified membrane proteins. A detergent-removal step then serves to promote the reformation of lipid vesicles and thereby encompass the proteins of interest within the vesicle structure. The initial solubilization of the lipid vesicles is performed by incubating them with a detergent that has a relatively high critical micelIe concentration. The lipid vesicles can represent ei:;ler a rr~xture of chloroform-methanol-extracted native membrane lipids, crude preparations of phospholipids (i.e., c ~ soybean phosphatidyicholine or asaleetin), mixtures of highly purified phospho|ipids (e.g. brain ph~phatidylethanoiamine, phosphatidyiserine and ph~phat~d~~o choline), or synthetic lipids (e.g., dimyristoy|-phasphatidylcholine). The more effective detergents for the initial solubilization step include 6ctyl glucoside, sodium cholate, sodium deoxycholate, or CHAPS. The incubation of the lipid vesicles with the sotubilizing detergent typically is performed for 30 ram-I h on ice. At this stage, the different protein components (e.g.. the /3-adrenergic receptor, G~, etc.) are added to the incubation. The proteins typically are not presen, during the entke detergent-lipid vesicle incubatio~ t'--~r~ since the detergents used to solubilize the lipid vesicles may have deleterious effects on receptors or G proteins. Once the protein components (which are usua:2 ia a variety of detergents, i.e., digitonin fgr the ~ ~_~r~~-

476 ergic receptor, Lubrol for G~), lipid vesicles, and solubilizing detergent are mixed together, the entire mixture is immediately subjected to the detergent-removal step. The removal of the detergents can be achieved by any number of procedures including dialysis, gel filtration and adsorption to detergent binding resins. We typically have favored the latter and have used Extracti-gel resin (Pierce) for these purposes since it is able to remove a number of detergents rapidly (within 30 rain)and yields a relatively homogeneous population of lipid vesicles which are about 2000 ~ in diameter. Typically ~ 0.5 ml of a mixture containing the solubilized phospholipid vesicles (1-2 mg/ml), the solubi|izing detergent (e.g., 0.8-1% octyl glucoside), the purified/3-adrenergic receptor (1-10 pmoi in 10-50 ~1 of 0.1% digitonin), and the purified G~ protein (1-50 pmol in 10-50 ~tl of 0.1-1% Lubrol PX) are added to I ml of Extracti-gel resin. ~ have found it to be advantageous to pre-equilibrate the Extracti-gel resin with bovine serum albumin (usually 2 mg/ml) in order to minimize the loss of receptors or G proteins during the detergent removal step due to the non-specific adsorption of these proteins to the resin. We have also obtained our best results (in terms of efficiency of incorporation of the receptors and G proteins into lipid vesicles) by equilibrating the Extracti-gel resin with buffers containing 10-20 mM MgC! 2. The presence of millimolar levels of MgCI 2 most likely accelerates the rate at which the phospholipid vesicles reform during detergent removal and therefore minimizes the time period during which the receptor and G proteins lack both detergents or lipids and thus are most susceptible to denaturation, aggregation, or non-specific adsorption to surfaces. Using the type of reconstitution p~tocoi outlined above, typically obtained lipid vesicles eluted from the Extracti.gel resin that contained about 1 ~-adrenergic receptor per vesicle were obtained. This ratio was determined by measuring the binding of radiolabeled antagonists to the receptor-containing lipid vesicles and by estimating the molar amount of vesicles present as determined using the known packing constraints for phospholipid molecules. In many cases, when reconstituting ~-adrenergic receptor/G~ (or/3-adrenergic receptor/Go/ adenylyi cyclase) interactions, it was found necessary to fuse these receptor/G~-containing vesicles together in order to improve the chances of a receptor in one lipid vesicle making contact with a G protein in another vesicle. This involved incubating the pmtein~ontaining vesicles that were eluted from the F~tractogel resin in polyethylene glycol (final concentration n 12.5%) for 5-10 rain at room temperature. The polyethylene glycol then was further diluted (by at least ten-fold) and the protein-containing vesicles were isolated by centrifugation at = 150000 x g for 1-1.5 h (c.f. Ref. 29). These aggregated vesicles worked very

well in assays of fl-adrenergic-stimulated activation of G~ or/3-adrenergic stimulation of adenylyl cyclase activity.

II-B. Assays of fl-adrenergic receptor / Gs interactions in reconstituted phospholipid vesicle systems The reconstitution of functional interactions between a hormone receptor and a {3 protein in lipid vesicles can be assessed by three different types of activities: (a) a high-affinity binding of the hormone to the receptor that is induced by the formation of a hormone/receptor/G protein complex; (b) a high-affinity binding of guanine nucleotides to the G protein which is the outcome of a hormone/receptorstimulated guanine nucleotide exchange reaction on the G protein; and (c) a hormone/receptor-stimulated GTPase activity in the {3 protein where this activity occurs as an outcome of hormone/receptor.stimulated GTP binding. The use of each of these assays in assessing the reconstitution of/~-adrenergic receptor/ G~ interactions will be described in the following sections below.

II.B. 1. High-affinity bi:Ming of agonists to receptors Binding experiments performed on plasma membrane preparations from various species have yielded agonist (isoproterenoD competition curves (i.e., competition between the agonist and a radiolabeled antagonist such as [ ~*l]iodocyanopindolol)that are indicative of two classes of binding sites [63]. These binding sites have been categorized as high-affinity (K d = 5-50 nM) and low-affinity (K d =0.1-5 /zM) and are felt to reflect the interactions of the agonist with a receptor-G protein complex, and with the free receptor, respectively. When the binding of radiolabeled antagonists to membrane preparations containing the ~-adrenergic receptor is measured, only a single class of antagonist sites is detected, i.e., antagonists do not distinguish between free receptors and receptor-G protein complexes. The addition of guanine nucleotides yields a single class of low-affinity agonist binding sites. This effect reflects the fact that guanine nucleotides cause the dissociation of the G protein from the receptor. Specifically, if sufficient guanine nucleotide is added to the binding assays, those (nucleotide-depleted) G proteins that are coupled to receptors will re-bind guanine nucleotide and then dissociate from the hormone/ receptor species, thereby returning the receptor to its low-affinity state for agonist. The demonstration of a G protein-induced high-affinity binding by agonists to the/3-adrenergic receptor, which is sensitive to the addition of guanine nucleotides, has been the most difficult of the receptor/G protein-coupled activities to achieve in reconstituted systems. Much of the difficulty probably stems from

477 orientation considerations. Most of the reconstitution protocols which have been used for co-inserting purified preparations of the fl-adrenergic receptor and G~ into lipid vesicles have yielded a mixed orientation of the proteins in the vesicles. Specifically, some of the G protein-coupling domains of the receptor proteins, as well as son e of the G proteins themselves, face the outside of the vesicle, while the remainder of the receptors (i.e., their (3 protein-coupling domains) and the G proteins face the inner vesicular space. Although the reconstituted phosphatidylcholine vesicles which are formed with the aid of octyl glucoside, cholate, or deoxycholate (diameters typically ranging from 500 to 5000 A,) are permeant to the more commonly used /3-adrenergic agonists (e.g., isoproterenol or epinephrine), they typically are not permeant to added guanine nueleotides. Thus, while the addition of hormone to lipid vesicles containing pure /3-adrenergic receptors and G, proteins can result in the saturation of the hormone binding sites of the reconstituted /3-adrenergic receptors, the addition of GTP or GTP-analogues to these vesicles may result in only a partial activation of the total pool of G~ proteins, i.e., those G~ proteins that are situated along the outer surface of the lipid vesicles. This then may result in a situation where only a portion of the total receptors that are coupled to G proteins in the lipid vesicles have their binding affinities for agonist influenced by added guanine nucleotide. Still, there have been a few examples where the reconstitution of G protein-induced high-affinity binding has been achieved in phospholipid vesicle systems. One such ease involved the reconstitution of the pure guinea pig lung/3-adrenergic receptor with the pure G, protein from human erythrocytes (c.f. Ref. 24). In these experiments, about 5 pmol of highly purified/3-adrenergic receptor (in a Tris buffer containing 0.1% digitonin) and 5 pmol of the purified human erythrocyte G~ protein (in a Hepes buffer containing levels of Lubrol ranging from 0.5 to 12%) were initially mixed with 1.7 mg of sonicated soybean phosphatidylcholine vesicles and 4 mg of octyl glucoside and the entire mixture was applied to a 1 ml Extracti-gel column. The protein-containing lipid vesicles that were eluted from the Extracti-gel were fused with polyethylene glycol and then isolated by eentrifugation. The resuspended vesicles contained 0.5-1 pmol (each) of fl-adrenergic receptor and G~ protein, i.e., roughly one receptor and one G protein per lipid vesicle. When these vesicles were examined for the ability of isoproterenol to compete for [~2Sl]iodocyanopindolol binding to the /3adrenergic receptor, the best results obtained from a number of such experiments indicated that initially - 30% of the/3-adrenergic receptor bound the agonist with high-affinity (Ka = 2 nM) and 70% of the total receptors were in a low-affinity state (K d = 300 nM). In

some cases, the addition of the non-hydrolyzable GTP analogue, GppNHp, was able to convert essentially 100% of the receptors to a low-affinity state, probably because these vesicles were leaky to the added guanine nucleotide. In other instances, the guanine nucleotideinduced conversion from high- to low-affinity binding was less complete. Thus far, it has not been possible to exploit this Gs-induced change in the binding affinity of the /3-adrenergic receptor for agonists as a means to monitor/3-adrenergic receptor/Gs coupling in reconstituted systems. As discussed below, isoproterenolstimulated GTP binding and isoproterenol-stimulated GTPase activity have served as more consistent readouts for these interactions. Before leaving this section, it should be pointed out that guanine nucleotide-sensitive high-affinity binding of agonists has been used successfully to examine the coupling of muscarinic acetylcholine receptors with different G proteins in phospholipid vesicle systems [37,38]. An excellent example of the use of this assay was provided by Florio and Sternweis [38]. These investigators found that membranes from bovine brain contained muscarinic acetylcholine receptors that bound agonists (e.g., oxotremorine with relatively low-affinity (K d = 1-10 #M) and that the binding of agonists was essentially insensitive to the addition of guanine nucleotides. However, when the receptors were solubilized from the membranes and then inserted into phospholipid vesicles, their affinity for agonists was significantly increased (i.e., the K d for oxotremorine = 0.03 #M) and the addition of guanine nucleotides caused a marked reduction in this binding affinity (i.e., the K d for oxotremorine was shifted to 10 #M). Thus these studies provide an interesting example of a case where the interactions between a receptor and an endogenous G protein were difficult to monitor in intact membrane preparations but could be readily assayed in reconstituted phospholipid vesicle systems. In these studies, the solubilized proteins were incorporated into the lipid vesicles by simply incubating the deoxycholate-extract (0.7% deoxycholate) with egg phosphatidylcholine (3 rag) and then removing the detergent by chromatography over a 50 ml Sephadex G50 column. The orientation of the incorporated receptors was not determined, although it is stated that 50% of the receptor binding sites measured in the original membrane preparation were present in the vesicle fractions. However, the data suggest that most, if not all, of the muscarinic receptors were able to undergo high-affinity binding in these vesicles and that the high-affinity binding could be completely eliminated upon the addition of guanine nucleotides (again suggestive that these vesicles were leaky to added nucleotide). An important question is why was the coupling between the brain muscarinic acetylcholine receptors and endogenous G proteins not initially observed in studies with mere-

478 brane preparations? One possible explanation is that the preparation of brain membranes resulted in some type of physical separation of the receptors from the G proteins and that this separation/constraint was overcome upon the co-solubilization of the receptors and G proteins and their co-insertion into lipid vesicles. Ion exchange chromatography was used to resolve the solubilized musearinic receptors from the G protein that was responsible for conferring the high-affinity binding of agonists [38]. When the resolved receptor was incorporated into the phosphatidylcholine vesicles, it demonstrated a low-affinity for oxotremorine (K a -l0 /.~M) with the binding being unaffected by added guanine nucleotide. However, when these receptorcontaining vesicles were incubated with the resolved G protein, and then subjected to G50 chromatography to achieve the co-insertion of the receptors with the G proteins into lipid vesicles, a high-affinity binding of oxotremorine (K d = 0.1 /~M) was regained. The a~dition of GTP then effected a complete reversal of the high-affinity binding back to a low-affinity binding state (K d -- 10/zM). Purified preparations of both the brain G i (probably a mixture of Git and Gi2) and Go proteins could substitute for the endogenous G protein in reconstituting high-affinity agonist binding to the muscarinic receptors, in the case, of the G o protein, this activity was partially reconstituted by the purified a 0 subunit (while the purified /3, preparation alone was incapable of reconstituting this activity) and fully reconstituted by a 0 together with /3v. In terms of the general mechanisms of receptor-G protein coupling, the results from these reconstitution studies provide some of the strongest evidence that G protein-induced effects on agonist binding are mediated by the a subunit of the G protein, with these effects then being accentuated by the/3 v subunit complex.

II-B.2. High-affinity binding by guanine nucleotides to reconstitated vesicles containing the [3.adrenergic receptor and Gs proteins Measurements of the hormonal-stimulated binding of radiolabeled guanine nucleotides to the G, protein, in two component lipid vesicle systems comprised of the purified ~l-adrenergic receptor and the purified G~ protein, have provided a sensitive monitor for receptor-G protein coupling. A variety of studies have been performed by Ross and colleagues characterizing the kinetics of guanine nueleotide binding [26,27,30]. In these studies, ~l-adrenergic receptors that were highly purified from turkey erythrocytes (in d,igitonin) and G~ proteins that were purified from rabbit liver (in Lubroi 12A9) were co-inserted into phospholipid vesicles by first incubating the receptors and the G~ proteins (in Lubrol 12A9) with dimyristoylphosphatidylcholine and polar lipids extracted t:rom turkey erythrocytes in a solution containing 0.36% deoxycholate and 0.04%

chelate. These detergents then were removed by Sephadex G50 chromatography, allowing the reformation of unilamellar vesicles which were about 1009 ,~ (average) in diameter with 1-4 receptors and 6-20 G, proteins per vesicle. ll-B.2.i. Correlation of the high-affinity binding of GTPyS and the activation of the Gs protein. Upon the reconstitution of the purified /3-adrenergic receptor with purified Gs proteins into phosphatidylcholine vesicles, the addition of hormone (e.g. isoproterenol) results in the stimulation of the high-affinity binding of [35S]GTPyS to the purified G~ protein. Using the types of reconstituted phospholipid vesicles described in the preceding section, Ross and his colleagues found that the agonist-bound /3-adrenergic receptor elicited an eight-fold increase in the extent of [3sS]GTPyS binding to G~ at early times (i.e., 1-5 min) and also elicited an increase in the rate of binding. These measurements were performed in parallel with assays measuring the ability of the reconstituted G~ to stimulate adenylyl cyclase activity, i.e., aliquots from the /3-adrenergic receptor/G~ vesicles were reconstituted with membranes from the murine lymphoma ($49) cell mutant designated cyc-. The time course for the activation of G~, as monitored by its ability to stimulate adenylyl cyclase in the eyc-membranes, and the time course for the binding of [asS]GTPyS, were essentially identical. These data clearly indicated that the/3-adrenergic receptor-stimulated high-affinity binding of GTPyS to reconstituted G~ directly reflected the functional activation of the G~ protein. II-B.2.ii. Dependence of the isoproterenol-stimulated [35SlGTPyS binding on the concentration of GTPyS. The isoproterenol-stimulated binding of GTPyS to G, in these reconstituted phospholipid vesicle systems was found to be half maximal at -~ 5 nM [26]. The binding data were consistent with a single :lass of high-affinity binding sites. As many as 8 G, p,roteins were stimulated to bind [3"~S]GTPyS by a ~ingle hormone/ receptor complex, consistent with the suggestions from membrane studies that the /3-adrenergic receptor can act catalytically to promote the activation of multiple Gs proteins (c.f. Refs. 63a, 63b). Increasing concentrations of GTPyS were found to increase both the initial rate of nucleotide binding to G, as well as the extent of binding [26]. The pseudofirst-order rate constant (K~pp) for the agonist-stimulated binding of GTPyS to the reconstituted Gs was found to be markedly influenced by the concentration of GTPyS. Specifically, at relatively low [GTPyS] ( --- 1 nM), the isoproterenol-stimulated value for K~pp was found to be only slightly greazer than that measured in the absence of agonist (--0.4 rain-~). However, with increasing concentrations of GTPyS, the value for K~pp for the isoproterenol-sfimulated GTPyS binding was increased by as much as ~en-fold. Plots of Kap~

479 versus [GTPyS] yielded a hyperbolic curve (rather than linear) dose-dependency, which suggested that the isoproterenol-stimulated binding of GTP3,S could not be described by a simple second-order bimolecular reaction (this will be considered in more detail below). 11-B.2.iii. Effects of Mg 2+ on the binding of ['~5S]GTPyS to reconstituted [3-adrenergic receptor / G~ t,esicles. The isoproterenol-stimulated binding of GTPTS to reconstituted G~ was potentiated by relatively low concentrations of free Mg 2+, i.e., the halfmaximal concentration for Mg 2+ was < 0.1 mM [26]. A similar concentration requirement was observed for the agonist-induced stimulation of adenylyl cyclase activity in cye-membranes. This suggested that Mg -'+ simply may serve to chelate the nucleotide, such that a metal-nucleotide complex would represent the active nucleotide species. At higher concentrations of Mg 2÷ (i.e., millimolar concentrations), a further increase in the total extent of GTPyS binding was observed, both in the presence and in the absence of isoproterenol. This, then, represented a receptor-independent stimulation of GTPyS binding by Mg 2+, probably as the outcome of a direct interaction between the divalent metal and the G~ protein. Unlike the case for the agonist-stimulated binding, the rate of the Mg 2+stimulated binding was independent of [GTPyS], suggesting that it was simply first order in G~. li-B.2.it'. Possible scheme describing the high-affinity binding of guanine nucleotides to G~. The results from reconstitution studies measuring isoprotere~,ol-stimulated [35S]GTPyS binding can be considered within the context of the following simple scheme (Fig. 1, also see Fig. 1 in Ref. 27). in the ca~e of the Mg-'÷-stimulated (receptor-independent) binding, where the rate con-

a-B-Y I

--

a +B'Y I

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a.B.y

"¢

,,

a

+B.y

7

GTPT'S

I

GTP)'S

GTPXS

4

I

GTP~'S

Fig. I. Scheme depicting the dissociation of GDP from the heteroirimetric G~ protein and the subsequent binding of GTPyS.

stant for GTPTS binding-ras found to be independent of [GTPTS], the rapid G';'P~,S binding reaction (reaction 5) may be preceded ":y a rate-limiting step such as the dissociation of the C.~ subunits (reaction 1) or the dissociation of GDP from the a subunit (reaction 6). The agonist-receptor complex would bind to the intact G protein and act to facilitate reactions 2 and 3 (see section II-B.2.~', below). Thus in the case of the agonist-stimulated binding of GTP3,S, the rate-limiting (first-order) step may be reaction 4. Ross and colleagues in fact proposed that reaction 4 reflected the size of the agonist stimulation and that the agonist-receptor complex may act by accelerating reaction 4 via the formation of a transient agonist-receptor-G,GTPTS complex [27]. ll-B.2-r. Role of GDP dissociation bt the agoniststimulated bhtding of GTP to G s. it typically has been suggested that the receptor-stimulated activation of a G protein is the direct outcome of a receptor-induced dissociation of a tightly bound molecule of GDP from the G protein (c.f. Refs. 64, 65). Since most purified G aroteins appear to contain nearly stoichiometric amounts of bound GDP, it is possible that the rate of GTP (or GTPyS) binding to G, might be limited by the rate of dissociation of the bound GDP. Brandt and Ross (c.f. Ref 30) have directly tested this possibility using recon~,titution approaches. Specifically, recept o r / G , vesicles were first allowed to exchange GDP for [3H]GDP m d then the lipid vesicles were washed and the release of [3H]GDP was directly compared with the binding of [35S]GTPyS. The results of these experiments indicated that the rate of agonist-stimulated [35S]GTPxS binding, subsequent to the agonist-stimuiated release of prebound [~H]GDP, was no faster than the rate of binding observed when both [3SS]GTPTS bindivg and ['~H]GDP dissociation were occurring concurrently. There was no immediate burst of GTPyS binding after GDP dissociation, and the rate of the agonist-stimulated dissociation of ['~H]GDP was actually twice as fast as the rate for the agonist-stimulated binrling of [35S]GTP'yS. Overall, these results suggested that the agonist-stimulated GDP dissociation is not rate-limiting for the agonist-stimulated GTP (or GTP~,S) binding eve-t. "A'laen considered in the light of Fig. 1, these results also suggest that in the presence of the agonist-receptor complex, reaction 2 occurs more rapidly than either reactions 3 or 4. May and Ross used reconstitution approaches to show that the /~-adrenergic receptor-stimulated binding of [35S]GTP~S to G., can be resolved into an initial, relatively slow event and if:on a diffusion-controlled0 second-order binding reaction [33]. When reconstituted phospholipid vesicle systems containing the /~-adrenel'gic receptor and the purified G, protein were preincubated with agonist, prior to the addition of GTP~,S, the agonist/receptor-stimulated binding was preceded

480 by an even more rapid burst of GTP-/S binding. The rate of the burst was second order in nucleotide and G~. Both the formation and the dissociation of the burst complex were found to be slower than the expected rates for the binding of agonist to the/3-adrenergic receptor, which then suggested the existence of a discrete step subsequent to hormone binding to the fi-adrenergic receptor. Based on studies that have already been considered (in sections i-iv above), it seems likely that the burst-phase represents reactions 2 and 7 in Fig. 1. This would be consistent with the finding that after preincubation, the agonist-stimulated binding is second-order with respect to [GTPyS] and appears to represent a diffusion-controlled process (i.e. steps 2 and 7 would occur during the pre-incubation period, enabling step 5 then to occur rapidly). On the other hand, in the absence of the pre,incubation of receptor and G~, the rate for the agonist-stimulated binding ~ows a saturable dependence on [GTPyS], since under these conditions, pathway 2-3-4 would predominate and reaction 4 would represent the rate-limiting step.

ll-B.3. Assays of hormone.stt;nulated GTPase activity The hydrolysis of bound G T P back to G D P (GTPase activity), which serves as a deactivation signal for G proteins [66,67], occurs as a natural outcome of the receptor-stimulated GDP-GTP exchange reaction and thus provides an alternative assay for the coupling of receptors to G proteins (c,f. Refs. 23, 24, 27. 30). A major advantage of this assay stems from the fact that the hormone-receptor-stimulated GTPase activity is part of a continuous (activation-deactivation) cycle of the G protein. The hormone-receptor-stimulated GTPas¢ typieMly occurs with turnover numbers of m, 1-4 min Ot, whereas the basal activity (i,e,, in the absence of hormone) often has turnover numbers as low as 0.01 rain = i, Thus, after 10-20 rain, a significant amount of •'ZPt can be generated in a hormone-stimulated manner, therelff providing a very sensitive indicator of receptor-G protein interactions, Ross and his col. leagues again were the first to report the use of this assay to monitor the coupling of the #-adrenergie receptor to the Gs protein in reconstituted phospholii~l vesicle s),stems [23], in their reconstituted systems, which consisted of the purified turkey erythro~t¢ ~-adrenergic receptor and the purified rabbit liver G~ protein, Brandt and Ross reported turnover numbers for the isoproterenol-stimulated GTPase activity of ~ 1 m i f t and found that these values were essentially the same as those measured for the isoproterenol-stimulated binding of [3sS]GTPyS. These workers also demonstrated that an agonist-re~ptor cornhad absolutely no effect on the rate of the actual GTP h~lmlytic event, indicating that the receptor only

influences the GDP-GTP exchange step and does not directly influence the deactivation step [30]. Due to the sensitivity of the receptor-stimulated GTPase activity, this assay has been used in a number of studies aimed at characterizing the relative abilities of different receptors (such as the ~-adrenergic receptor, the a2-adrenergic receptor, rhodopsin) to interact with different G proteins, e.g., preparations of the human erythrocyte G~ protein, the human erythrocyte G i protein (mainly Gin), the brain G i proteins (mainly a combination of Gil and Gi2), the brain G, protein, and bovine retinal transduci~ (c.f. Refs, 23, 24, 29, 30, 34, 36). As expected, the fl-adrenergic receptor showed a high degree of selectivity for the human erythrocyte O~ protein over the G i protein preparations, or transducin, in these reconstitution studies [29]. it was found that the fold stimulation by isoproterenol of the GTPase activity of the G~ protein could be accounted for by the hormonal-stimulations of the rate and extent of [a'sS]GTP~,S binding to G~ [29]. The increase in the extent of GTP,/S binding may be mainly attributed to an increase in the overall stability of the G~ protein due to the coupling of G, to agonist-receptor complexes (c.f. Ref. 26). The increase in the rate of GTP~,S binding most certainly reflects the facilitation of a conformational change in the G~ proteitl which either enhances GDP dissociation and/or the association of GTP~S (as already discussed above; also c.f. Ref. 30). With regard to ~8.adrenergic receptor-G i interactions, studies with the mammalian receptor and the human erythrocyte G i (Gin) protein suggested very little tendency of the /]-receptor to effectively couple to this form of G4 [29]; however, studies with the avian //adrenergic receptor and the rabbit liver G l protein (where the majority of the preparation was probably Ga) suggested a much more significant level of reactivity [68]. The physiological significance of this interaction remains to be determined. Reconstitution studies also have been performed to directly compare the abilities of the purified human platelet a,-adrenergic receptor and bovine retinal rhodopsin to interact with different GTP-binding proteins [36]. The results of these studies suggested that the •,adrenergic receptor was able to effectively couple to the human erythrocyte Gi protein (i.e., Gi3), the bovine brain G~ proteins (i.e., G a and G~z), and the brain Go protein. The maximum turnover numbers for the ¢~2-adrenergic receptor-stimulated GTPase activities of the different Gi and Go proteins were similar to those measured for the /3-adrenergic receptor-stimulated GTPase activity of the Gs protein (i.e., = 1 reel 32Pi released per rain per reel G protein). As expected, the az-adrenergic receptor showed little ability to promote the activation, or subsequent GTPase activity, of the Gs protein. However, somewhat unexpected was the find/ng that the reconstituted a2-adrenergie recep-

48t tor was unable to effectively activate the retinal GTPbinding protein, transducin. The turnover numbers for the a_,-adrenergic-stimulated GTPase activity of transducin typically were 10-25% of those measured for the G i and G~ proteins. This finding was especially surprising givt, a the fact that the primary amino acid sequence of the transducin protein is much more similar to the G i and G, proteins than to G~, and that the carboxyl terminus of transducin (which contains the putative receptor-binding domain) shows a high degree of similarity ~o the earboxyl terminii of the G~ and G, proteins. I hus, these results suggest that there are additional regtons on the G protein-a subunits, i.e., aside from the extreme carboxyl terminal amino acids, that are involved in coupling to receptors. Unlike the ~.,adrenergic receptor, rhodopsin was less discriminating in its interactions with the different G proteins, i.e., the photoreceptor appeared to interact as effectively with the G, and G, proteins as with tran,,:ducin [3(~]

H.C Use oi" reconstittaion and molecuh : biology approaches to map key domains bu,oh,ed in receptor / G protein #~teractions H-C 1. Receptor studies in recent years there has bee,~ a great deal of success in the isolation of cDNAs for the/3-adrenergic receptor and related hormone receptors as well as for different members of the G protein family [69-82]. The development of appropriate expression systems for these cDNAs have provided a novel and relatively rapid approach for the identification of domains on the receptors and G proteins that are essential for the

effective coupling of these protein components. The limited tryptic digestmn of receptor proteins has also been employed in conjunction with reconstitution studies as a means of identifying regions on the receptors that ate essential for coupling to G proteins. The types of results that have been obtained from these different studies will be described in more detail below. lI-Cl.i. Molecular biology studies. The complete amino acid sequences for the mammalian fl~- and /~,-adrenergic receptors, the avian/~radrenergic recep, tot, and a number of other receptors that interact with G proteins including the mammalian a r and a2-adrenergic receptors, the muscarinic acetylcholine receptors (MI-M5), and rhodopsin have been determined [69-76,83]. All of these receptors share sequence and topo° graphical similarities with each other. Each of these proteins are comprised of an extraceilular domain that contains a number of potential sites for N-iinked glycosylatlon, seven transmembranal helical segments, and a cytoplasmic domain composed of three shor~ loops (10-30 amino acids each) and a carboxyl terminal tail, that in many cases contains a number of potential sites for (regulatory) serine phosphorylations as well as possible sites for acylation [84]. The proposed membrane arrangements of the /~,,-adrenergie receptor, and the photoreceptor, rhodopsin, are depicted in Fig. 2. Hormone bhtding domah~. A number of lines of evidence have led to the suggestion that adrenergic ligands bind to amino acid residues that arc present within the putative transmemb~anal helices, rather than to regions located within the extracellular hydrophilic domain. These suggestions would be in line with the situation for rhodopsin where the membrane-spanning 20

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Fig. 2. Postulated membrane-arrangement of the/~-adrenergic receptor and opsin. In the case of the ~oadrencrgic receptor, the .umber~ correspond to the following amino acids: phenylalanine 10. prolin¢ 20. glutamic uckl 30. lysine 60. lyrosin¢ 70. aspuragine 1000 ly~ine !40. ly~i,e 180. cysteine 190. leucine 2.'40.phenylalanine 240. glutamine 251). arginin¢ 2(X). lysine 27t). asparlic acid 3tg). leucine 34(L tyrosi,e 350° glyci,e 3¢~0. glycine 370. glutamic acid 380. glutamie acid 390 and proline 41}0. in the case of rhodopsin, the ,umbers correspond to the following amino acid~: tyrosine 10. valine 20. tyrosine 3l). throonine 7(). histidine l(10. cysteine 110. glutamic acid 150. aspartic acid 100. valine 23(I. scrine 2411.v~din~:25()~ glycine 280. threoninc 320. aspartic acid 330. and throonine 340. Although not depicted here. it has been suggested that the acylatio, of a cysteine residue in the carboxyl terminal domain actually causes the carboxyl terminal to associate with the membrane, thereby creating a fim~fh cytoplasmic loop (c.f. Ref. 84).

482 helices have been suggested to form a binding pocket for the chromophoric moiety, retinal (with the specific point of attachment being a Schiff base linkage between retinal and the lysine at position 296 in opsin). Dixon, Strader and their colleagues have examined the question of the location of the hormone binding domain on the/32-adrenergic receptor by expressing the cDNAs for the wild-type hamster ~2-adrenergic receptor and a series of deletion mutant genes for this receptor in mammalian cells (i.e., COS-7 or L cells) [85-87]. Two such mutants were found to differ significantly from the wild type receptor in their abilities to bind ligands [871. One of these, which lacked residues 274=330, i.e., the region encompassing the transmembrahe helices V! and VII, was incapable of binding either agonists or antagonists, while another mutant, which lacked residues 179-187 from the third external segment, bound antagonists and agonists with reduced affinities, Neither the deletion of the amino terminus (residues 21-~1), the removal of 65 amino acids from the earbo~l terminus, nor the elimination of the third cytoplasmic loop (i,e,, the deletion of residues 229-236, or the deletion of residues 239-272) had any effects on ligand binding. However, the mutant which lacked residues 239-272 was not capable of elieiting an isoproterenol-stimulation of adenylyl cyclase activity, which suggests that this region may be involved in coupling the receptor to the G, protein (see below), ~xon, Strader and their colleagues also examined whether conserved negatively charged residues, that are located within the hydrophobic membrane-spanning regions of the #-adrenorgic receptor, might be directly involved in ligand binding interactions [85], Specifically, there are a few conserved aspartate and glutamate residues within the membrane-spanning heIkes of the B-adrenergie receptor that could be involved in interactions with the positively charged amino grou~ on the adrenergic ligands, One l~3~sible candidate, aSl~rl~te 79, is located within the second lWdrophobic region and is conserved in all known B" adrenergic r~eptor s,.-~iuences, the muscarinie acetylcholi~ receptors, and rhodopsin, A second candidate, glutamate 107, is conserved among the #-adrenergic r e c e i p t sequences (there is an aspartate residue at an analogous position in the mu~arinic acetylcholine receptor), Other candidates included aspartate 113, which is censored among the/]-adrenergic receptors and the muscarinic acetylcholine receptors (but not rhodopsin), a~vJ asparagi~ 318, which is conserved among the sequenoes of hormone receptors and which occurs at a position analogous to the iysine 296 in opsin, Dixon, Strader and coworkers found that the replacement of a.spartate 79 and asparagine 318 resulted in receptors that were able to bind antagonists with affinities similar to the native receptor but bound agonists with

much weaker affinities. The replacement of the alanine at position 107 had no obvious effects on ligand binding to the receptor. However, the substitution of an aspartate residue at position 113 completely eliminated ligand binding to the receptor. This led to the suggestion that the binding of adrenergic ligands to the fl-adrenergic receptor may involve a hydrogen bond between the carboxylate group of aspartate 113 and the p.'otonated amino group of the ligand, G p~tein binding domah~, As indicated above, the deletion of residues 239-272 from the fl2-adrenergic receptor resulted in a total loss of isoproterenolstimulated adenylyi cyclase activity. While the deletion of residues 229-236, 238-251, and 250-259 had no effect on the isoproterenol-stimulated activity, the deletion of residues 258-270 resulted in the attenuation of this activity and the deletion of residues 222-229 resulted in the complete Io~s of the agonist,stimulated activity [87]. These results led to the suggestion that within the third cytoplasmic loop, tile peptide segments forming the junctions with both adjacent hydrophobic domains were required for coupling to the G, protein and eliciting a stimulation of adenylyl cyclase activity. Lefkowitz, Caron and their colleagues took a somewhat different approach to identifying domains on tile /]-adrenergic receptor that were essential for coupling to GTP-binding proteins (c.f. Ref. 88). Rather than making interpretations based on the loss of functions that accompany mutagenesis, they set out to identify ~sitive functions associated with specific receptor domains following the expression of a variety of chimeric a,- ~-adrenergic receptor genes. Ten chimeric receptor genes from the human /],-adrenergic receptor and the human platelet a,-adrenergic receptor were construeted (Fig, 3) and expre~ed in Xenopus o¢~'ytes and COS-? cells, and then the abilities of the chimeric receptors to bind /],-adrenergie- and a,-adrenergicspecific ligands, and to activate adenylyl eye!use, were determined, The chimeric receptors CR8 and CR9 (FLg, 3B) were found to contain the shortest stretches of the B~-adrenergie receptor that can activate adenylyl cyclase activity. The CR9 was less efficient in stimulating cyclase activity than CR8 as indicated by the halfmaximal concentration for agonists and the maximal stimulation of adenylyl ~clase activity. The CR3, which contains the/~,-adrenergic amino acid sequences corresponding to tile sixth and seventh membrane-spanning helices and the earboxyl terminal tail, did not elicit the stimulation of adenylyl cyclase activity. Thus, the results of these chimera studies suggests that the third cytoplasmic loop and portions of the fifth and sixth hydrophobic domains may be involved in the coupling of the g2-adrenergic receptor to the G, protein, while the membrane-spanning helices 1, 2, 3, 4 and 7, as well as the first and second cytoplasmic loops

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Fig. 3. Post,hired m e m b r a n e arrangements of the /:L," and ~L, ~ldrenergic reeepiors. A: poslnlated :aructures ~i Ih~ w~ld type c~2- and /],,-adrenerllic receptors. B: postulated structures of ~z2/#,-adrenergie recepit)r J2hillleras. The construction (fl" the different chimeras was performed as described in detail by Kobilka el al. [70]. Portions ~f this figure also were t:lken from Kobik~t et al. [70].

and the carboxyl terminal tail, may have little influence in determining the specificity of coupling of the B~,adrenergic receptor to G,. Lefkowitz, Caron and their colleagues extended these studies by examining the effects of multiple mutations of the amino acids in the cytoplasmic domains of the human /32-adrenergic receptor [89]. The results of mutations in the carboxyl-terminal segment of the major cytoplasmic loop of the /32-adrenergic receptor indicated that amino acid residues in this portion of the receptor may play a role in receptor/G protein coupling. None of the mutations of this region corn° pletely abo,shed the receptor-mediated activation of adenylyl cyclase, which suggested the possible involvement of other regions on the receptor in coupling to the G, protein. Two other regions of the receptor that were implicated in coupling to the G, protein were the

amino terminal segment of the c~opla.,,mic tail and the second cytoplasmic loop. Changes in ~rnino acids 327°~ 330, and 330--334, resulted in 30-40% reductions in the efficacy of the isoproterenol-stimulated adenylyl cyclase activity; simil~!~,~, the substitution of cysteine 341 with glycine resulted in = 41)% reduction in the ability of the reeeplor to stimulate adenylyl cyelase activity. The substitution of the h;~hly conserved proline residue at position 138 (fi,~ threonine) .~,;hin the second cytoplasmic loop of the receptor als,)~resulted in an approx, six-fold reduction h! the '.abi!i~:, of isoproterenol to stimulate adenylyi cyelase actiw'y. Over° all, these investigators postulated that the ami~.~olermio nal portion of the carboxyl-terminal tail play:~ a direct role in coupling to the G~ protein. It w:t~, further suggested that cysteine 341, within the carboxyl terminal ta ', and the region of the second cytoplasmic loop

(and in particular, proline 138) may play a supportive role in reccptor/G protein coupling by maintaining the proper configuration of the amino-terminal segment of the cytoplasmic tail in relation to th~ carboxyl-terminal portion of the m ~ r cytoplasmic loop. More recently Ross and coworkers took a similar approach to obtain information about the O~-coupiing domain on the ~ 3 r e n e r g i c receptor by substituting cytoplasmic domains of the turkey/]:-adrenergic receptor for the corresponding regions of the M l-mu~arinie ebolinergic receptor (Refs. 89a. 89b), "['he replacement of the large third intracellular k~op of the mu~arinie cholinergic receptor with the homologous ~gment of the/]oadrenergic receptor conf~rred the ability to regulate O~ (and thereby ~timulate ~;dcnylyl cyclase activity) in the membrane fractions ~ff COS, A293, or S, [rug~rda (Sf'4pcells without al~,tishing the mu~;arinic(~nd (}TP-) ~pendent regu!~tion of phosphatidylinositol lipid turnover. Interestingly, the d~ccapeptide from the a m i ~ terminal portion of the third intracellular loop of the /J-adrenergie receptor was sufficient to elicit the activation of (}~ and the stimulation of adenylyl cyelase by an otherwise unaltered MImusearinic cholinergic receptor. This then suggests that the amino terminal portion of the third intrucellular loop of the/]-adrenergic receptor forms at least part of the binding domain for the G~ protein, It was ,~peculated by these authors that this region of the/~l-adrenergic receptor also may play a role in catalyzing guania¢ nuclcotide exchange on G,, given the fact that the structure of the amino terminal region of the third intraccllular loop of the/~-adrenergic receptor is predicted to form an amphiphilic a-helix similar to the peptide mastoparan, which is a known activator of G proteins [89c], An additional pie¢~ of information which cam~ from thi~ work was the finding that the second intracellular loop ot' the #~adrenergi¢ receptor may play a role in ~ g u l a t i ~ G protein-selectivity, Specifically, the replacement of the second intracellular loop of the MImtr~adnie eholinergic receptor (with that of the turkey Bl-adrencrgi¢ receptor) altered sel~tivity by diminishin8 the muscarinic cholin~rgic stimulation of phosphoimJedtide tumo~r, without markedly enhancing the stimulation of ~denylyl cTelase, Thus it was proposed that the third intr~c~llular loop probably interacts functionally with the second intracellular loop to ensur~ that the (3~ p ~ e i n is stimulated by the ~-adren¢~ie r~ptors, Ho~ver, this may not b~ the ease for all G protein-link~:~ receptors since other studies with the MI- and M2-mu~v,dnie eholinergie receptors suggest that the third intra~llular loop may have a ~aominant role in determining G protein selectivity [89d], ~I-C.LiL To~pti¢digestion studies. The limited digestion by tpjpsin of the/~-adrenergie receptor, and the photore~ptor, rbodopsin, has also been used to obtain

information regarding the nature of the receptor-domains involved in receptor-G protein coupling. Work by Litman, Hargrave and colleagues demonstrated that the proteolytic removal of the small carboxyl-terminal tail of rhodopsin is not required for the light-stimulated activation of the retinal GTP-binding protein, transducin [90,91]. A single tryptie cleavage within the third intraeellular loop of rhodopsin a l ~ did not adv e r i l y affect the light-stimulated activation of transducin, whereas more extensive proteolysis of this loop had deleterious effects on rh(~lopsin-transducin coupling. The~ latter results were in general agreement with the results obtained fi'om molecular biology studies of the /J-adrenergic receptor (see above) and rh~lopsin (c.f. Ref. 91a) which suggested that ptwtions of the third cytoplasmic It~t~pof the receptor may play an important role in mediating the regulation of the G~ protein. Ross and his colleagues performed a careful exami° nation of the ability of the trypsin.treated lJcadren. ergic receptor, purified from turkey erythrocytes, to couple to the G, prote~n usmg reconstitution approaches [92]. These hwestigators round that the treatment of the avian /3cadrenergic receptor with high concentrations of trypsin did not result in the c~mplete proteolysis of the receptor. The limit digest contained a silver-stained band with a mobility of M,-- 19000 ldesignated as fragment A) and a second band with a M~ ~ 11000 (designated as fragment B). This pattern ap~ars to ~ similar to that displayed by rhodopsin. The amino-terminal sequence of fragment A was determined to begin at glutamine 30 of the avian /~c adrenergic receptor sequence and to include at least the four amino-terminal membrane-spanning domains and the intervening loops and possibly the fifth membrane-spanning region. Fragment B was suggested to begin at vaUne 280 within the m~jor intrace!!ular loop and to include the two earbo~l-terminal membranespanning sequences and the intervening extraeellular loop, but a minimal amount of the intrace!iular car. boxyl-terminal region (e.f. Ref. 92). Affinity chromato. graphic repurification of the trypsin-treated receptor resulted in the isolation of both fragments A and B; however, the major cytoplasmic loop did not co-purify with these fragments. The ability of these fragments to mediate the activation of the G, protein was assessed following their co-reconstitution with G~ into phospholipid vesicles. Isoproterenol was found to stimulate the binding of GTPTS to G,, in lipid vesicles containing the tryptic fragments of the avian/~cadrenergic receptor, to a similar rate and extent as that observed with lipid vesicles containing the native receptor. The limittryptic fragments of the /3-adrenergic receptor also retained their ability to be stimulated upon reduction by dithiothreitol (e.f. Ref. 93). Thus, these results appeared to rule out the hydrophilie earboxyl-terminal

485 tail and most of the largest intracellui,. (third) hydrophilic loop as being directly involved in the regulation of G~. At first glance, these findings seemed to contradict those from molecular biology studies which suggested that the third cytoplasmic loop confabbed essential amino acids for mediating G protein coupling and regulation. However, it is possible that those regions removed by the limited trypsin treatment of the avian/Jt-adrenergic receptor do not represent the portions of the third cytoplasmic loop (i.e°, the aminoterminal- and carboxyl-terminal-segments) that were implicated in the coupling of the mammalian B,-adrenergic receptor to G,. Another possibility is that the deletion of amino acids by molecular biology approaches results in coat~rmational alterations that have deleterious effects on reeeptor~G protein coupling, Fnlure studies will require a careful analysis of |his queslion through the use of a varie|y af approaches, in¢lud.o ing molecular biological and protcht chemical ~protcol° ysis studies) approaches Iogether with the use of differ° eat synthetic peptide segments h'om the receptor.

H-CZ G proteins A number of structure-function studies of the G~ protein have been performed using molecular biologybased approaches. For example, Bourne and his colleagues demonstrated that the extreme carboxylterminus of the ,~ subunit must be involved in the coupling of G~ to the/J-adrenergic receptor, based on the finding that in a mutant mouse $49 lymphoma cell line (uric), in which G, cannot be activated by hormone receptors [94], a point mutation in the a~ earboxyl terminal tail substitutes a prolinc residue for an arginine. These data were consistent with the findings from other biochemical studies which suggested that the earbo~l terminus of G protein.a subunits were impor~ tant for binding to receptors (c.f. Ref. 95). Based on the conservation of primary structure among the different G protein-a subunits, Masters, Stroud and Bourne proposed a topographical model for a composite G protein-a subunit [95]. The composite a subunit was divided into three domains: domain ! was suggested to be responsible for binding the fly subunit complex, domain I1 was proposed to contain the region essential for interacting with effeetors, and domain I!I was suggested to be responsible for directly binding receptor proteins. Masters et al. set out to test this model by constructing a chimeric a~/a~ cDNA which encoded a polypeptide composed of the amino terminal 61)% of the a~2 chain and the carboxyl terminal 40% of a.~ [96]. This chimera was introduced into $49 cyc- cells, which lack endogenous a,, with the expectation that it would be incapable of mediating hormonal stimulation of adenylyl eyelase activity, i.e., because the presumed effector domain was comprised of the ai2 subunit sequences rather than a.~ sequences.

However. these investigators obtained the unexpecl~.d result that the a,,/a~ chimeras were able to effectively stimulate adenylyl cydase aclivity in a /J-adrenergic receptor-promoted manner. These findings indicated tha! the carboxy[ terminal amino acid sequences of a~ (i.e., domain Ill) contain the necessary structural fealures |'or mediating the specific stimulatory coupling of the fl-adrenergic receptor to adenylyl cyclase. Johnson and his colleagues have used similar approaches to obtain information regarding the domains responsible for the regulation of a~ function [97-100]. Based on the results from studies examining a number of types of a~/a~ chimeras, these investigators proposed that the adenylyl cyclase activation domain on the a~ subunit lies within residues !le235-Gly355, with the unique a~ sequence (Thr325oArg336) being essenlial i~r the stimulation of adenylyi cyclase activity, and sequences within the amino terminal domain of a, being responsible h)r regulating the rate of activation of this subunit by guanine nucleotides. Regarding the latter point, i! was further proposed that the a, sequence within residues Glul5 to Pro144 represented an attenuator control domain, i.e., controlling the rate of GDP-GTP exchange. These regulatory properties were suggested to possibly involve/~, interactions with a~. For example, the ability of fly to inhibit GDP dissociation, and to attenuate the activation of adeny[yl cyclase activation, is lost in a~ chimeric subunits which contain the first 54 (or 64) amino acid residues of a~, and these a,2/a ~ chimeras show constitutive stimulation of adenylyl cyclasc activity. Overall, the results of the various molecular biology-based studies of the a~ subunit have led to the identification of five regulatory domains; the~e are (moving l)'om the amino terminus in the direction of the carboxyl terminus of the polypeptide) the attenuator domain, the GAP region (which is responsible |o~ stimulating the intrinsic GTPase activity of a,), the GTPase region, the effector-binding domain, and the receptor-binding domain. In the future, the combination of these molecular biology-based strategies with reconstitution approaches using purified protein como ponents should prtwide a detailed structure~function picture of the G protein-a subunit in its role as a signal transducer.

IlL Rhodopsin/ transducln system The rhodopsin-coup!ed vertebrate vision system represents another example of a receptor-G protei~°couo pied signaling system that has been extensively studied. in this ease, the signaling pathway is initiated by the absorption of light by the photoreceptor, rhodopsin, Rhodopsin is comprised of a polypeptide backbone, opsin, which is a glycoprotein of M r --4001_10 and which appears to be comprised of seven transmem-

brahe helices. The chromophoric moiety, retinal, is attached by a Schiff base linkage to a lysine residue at ~ t i o n 296 in the seventh membrane-spanning region. The absorption of light by this chromophore serves to activate opsin, presumably in a manner that is similar to the activation of the ~-adrenergl¢ receptor upon the binding ~ spech~ic agonists. This activation results in the effective coupling of rbodopsin to the GTP.binding inotein, transducin. The structure of the retinal G protein is similar to the structures of the G proteins (G~ and G i) involved in the honaonal regulation of edenylyl cyclase. Specifica,y, tran~do¢in is heterotrimeric, with its subunits desiiInated as aT (Mr ~ 3 9 ~ ) , / ~ (Mr ~ 36000), and Y~r (M~ ~ $-100(~)), The 36 kDa 0 subunit of transducin at~ea~ to be highly similar, if not identical, to one form of the 0 subuult (i.e., commonly designated a s / t ~ ) that is utili~d by the G~ and G, proteins. On the other hand, the ax and YT subunits are structurally distinct from the a and y subunits of the other members of the heterotrimeric G protein family, The effcctor enryme in the vertebrate vision system is the cyclic GMP phosphodiesterase (PDE). This endiffers significantly from the known structures of other biological effectors for G proteins such as adenylyl ¢yclase, the phospholipase enzymes, and various ion cbannels such as K* channels and Ca;* channels. The PDE is made up of three types of subonits, Two of are designated as ¢ and/3 (i.e., tPD~, 0 ~ ) and have molecular weijhts in the rang~ of 85-90000, The third subunit, designatc¢l y (ypDl~)has a Mr of 14000, Recent studies sug~st that the PDE molecule has a sobunit stoiehiomet~ of 1 opec, 1 ,Bpt~t~, and 2 YeD~ subunits [101], It has been s ~ s t e d that the stimulation of PDE ~.'tk'!ty occurs by the removal of the Ye~ subunits from the ¢o~ of the e n ~ a ¢ [102-10S], IAmited trypsin t ~ t m e n t of the cyclic GMP PDE results in the .selective dit~Mi~ of the y ~ sutmnits and this is accom. Imaied by a maximal stimulation of cyclic GMP hydrolysis by the ems~ne. The addition of isolated ~'po~ sa~nits to the tcy~in-~ctivated enzyme then results in an inhibition of en~nne ~¢tivity, Based on these results, it has always been assumed that the activated G protein, transde~n, must bind to the YPDi~subunits and causes their dissociation from the larger molecular weil~ht (a~os and , 8 ~ ) subunits. The hydrolysis of the bound GTP to GDP by eT has been presumed to re~lt in the release of the exfGDP) species from the y ~ ~lbuni~ ~ J l ~ ulk~vin~ the VPDE to rcassociate ~ the larger molecular weight subenits of the and the ~x(GDP) to rcassociate with the #~T sebenit ¢Oml~eXof transducin. With the recent realization that there are two , y ~ subunits per enzyme molem~ [101], questions have arisen regarding whether ¢~e or two aT(GTP) species are necessary for full

stimulation of enzyme activity and/or if the binding of two aT(GTP) complexes per enzyme result in a disproportionately higher extent of enzyme activity than that measured when one aT(GTP) binds to the enzyme. IliA. Reconstitution studies / GTPase measurements

Bernard Fung was the first to investigate the interactions of rhodopsin with transducin in reconstituted phospholipid vesicle systems [34]. In these studies, purified rhodopsin was mixed with egg yolk phosphatidylcholine and egg yolk phosphatidylethanolamine at ratios of | : I ~ (protein:lipid). The incorporation of rl'~xlol~sin into the phospholipids was achieved by extensive dialysis. In these reconstitution studies, it was possible to directly add the transducin subunits (c~T and /J3'T) tO the rhodopsin-containing lipid vesicles since both the aT and flYX subunits were purified and stored in the absence of detergent. Thus, the receptorO protein interactions which were assayed in these systems represented the interactions occurring between the added G protein subunits and those rhodopsin molecules whose G protein-binding domains were facing the extravesicular space. bung used the light (rhodopsin~-stimu!ated GTPase activity as a monitor for receptor-G protein coupling. He observed that an assay incubation which contained only the a T subunit and photolyzed rhodopsin exhibited low GTPase activity (i.e., turnover numbers of 0.1-0.2 rain-I). The addition of the /31,T subunit complex then caused as much as a 40.fold promotion in the rates of GTPase, with the activity being linearly proportional to the a T subunit concentration at all ratios of ~T to OTT (these ratios were varied from roughly I : 1 to 15:1). When the rhodopsin-stimulated GTPase activity was examined as a function of the concentration of the O~'x subunit complex, the activity was observed to saturate at levels of/3YT which were substoiehiometric relative to the aT subunit, i.e., halfmaximal GTPase activity occurred at a ratio of a T: ~YT of 20-25 : 1. These results indicated that the/$YT sub. unit complex was able to recycle and act catalytically in promoting the rhodopsin-stimulated GDP-GTP exchange and GTPase activities within the a T subunit. These findings also confirmed earlier suggestions that the Oy subunit complexes must dissociate from their a subonits following the activation event (53-55). lll-B. Fluorescence studies

Recently, we have made use of fluorescence spectroscopic techniques in conjunction with steady-state kinetic approaches to examine the rhodopsin-stimulated activation-deactivation cycle of transducin in reconstituted phospholipid vesicle systems [106-107]. The intrinsic tryptophan fluorescence of the a subunits of

487 G proteins can be used to distinguish between their GDP- and GTP-bound states (c.f. Refs. 106-107). Specifically, the direct comparison between the intrinsic protein fluorescence of the pure aTGDP subunit and the pure a-rGTPyS complex illustrates that there are 2-2.54old differences in the fluorescence emission of these species with the aTGTPTS complex showing the higher emission (Fig. 4) (also see Refs. 106, 107). A similar enhancement of the intrinsic tryptophan fluorescence occurs when AIF4- is added to the aTGDP complex (Fig. 4). These results suggest that the conformational change in the a T subunit, which accompanies its activation, alters the local environment of one or both tryptophan residues on a T. Essentially the same results have been reported for both the a o and a i subunits by Higashijima and colleagues [108,109]. In the case of transducin, ,;hanges in intrinsic fluorescence can be used to directly monitor the receptorstimulated GTP binding-GTPasc cycle of the G protein in real time. For example, when the pure aTGDP complex is added directly to a solution of the ~ Y T subunit complex and phosphatidylcholine vesicles containing light-activated rhodopsin, the addition of GTPyS elicits an immediate enhancement in the tryptophan fluorescence reflecting the formation of the aTGTPyS species (Fig. 5). The addition of GTP, but not GDP, to these reconstituted systems also induces a rapid enhancement in the tryptophan emission, although this enhancement is accompanied by a slow relaxation of the fluorescence back towards its original basal level. There are a number of lines of evidence which indicate that the fluorescence decay, which follows the rhodopsin- and GTP-induced fluorescence enhancements, directly reflects GTP hydrolysis within

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Fig. 4. Intrinsic fluorescence of the purified aTGDP and aTGTPyS complexes of transducin. The aTGDP and aTGTPyS complexes were purified from bovine retinal rod outer segments as described in Ref. 106. The fluorescence emission spectra for aTGDP (100 nM) and aTGTPTS (fl00 nM) in 100 mM Tris-HCI (pH 7.5), 25 rrM MgCI 2, and in the presence of t0 mM NaF, 10 p.M AICI 3 were scanned while exciting at 280 nM.

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I00 sac Fig. 5. Rhodopsin and GTP-stimulated enhancements of the intrinsic fluorescence of the a T subunit. Rhodopsin, the a T G D P complex, and the H YT subunit complex were purified as described in Reg. 10t} and the rhodopsin was inserted into phospholipid vesicles as outlined. A mixture of rhodopsin-containing lipid vesicles ([rhodopsin] = 30 nM), /~')'T (130 nM), and a T G D P (200 nM) was incubated in room light for 10 rain at room temperature. GTP, or GTPyS, was added to the final indicated concentrations, and the change in fluorescence emission (excitation = 280 nm, emission = 335 rim) was continuously monitored. These data were taken from Guy et al.

B00l. the a T subunit. One line of evidence is that increasing the concentrations of GTP causes the fluorescence enhancement to persist for longer periods of time. This is the expected result if the fluorescence decay in fact reflects GTP hydrolysis, since increasing concentrations of GTP should cause the a T subt;nits to remain in a GTP-bound state for an extended period of time, thereby extending the period of enhanced fluorescence. In addition, the integration of the time-dependent fluorescence changes corresponds closely with the rate of [732P]GTP hydrolysis (c.f. Ref. 107). This, again, would be expected if the fluorescence enhancement were due to the formation of an activated GTP-bound a T subunit, which undergoes a first-order reaction (GTP hydrolysis) to yield Pr III-B.1. Changes in a r fluorescence as a function of [rhodopsin] and [GTP] Both the rate and the extent of the GTP-induced enhancements in the a T fluorescence were dependent on [rhodopsin], whereas only the rate (and not the extent) of the GTPyS-induced enhancements were dependent on the levels of the photoreceptor (c.f, Ref. 107). The net fluorescence enhancements elicited by the addition of GTP represented a composite of the conformational changes within the aT subunit which accompanied GTP bindirg (i.e., fluorescence enhance-

488

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GTP

kr~vR'(I(i,, )~ind R.(~(GDF)(

a(GDP) (

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khyd .~-

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Fig, 6, Model for the activation-deactivation cycle of the a T subunit. Keul represents the rate constant for the rhodopsin-stimulated GDP dissociation from a(GDP). Within this scheme only the rhodopsincoupled a(GDP) species is able to undergo guanine nucleotide (GDP-GTP) exchange over the time p=riod of our assays (i.e., Kr~I is extremely slow in the free a(GDP) species and thus is not shown to occur), The OTP-binding event (Kbiad) is assumed to be a himolecular reaction between R e ( ) , the nucleotide-depleted

rhodol~in-cT complex,and GTP which results in the conversionof aT tO an active conformation (or*). /3yT is required for the rhodopsin-stimulated guanine nucleotideexchangereaction on aT; however, for simplicity,the/3yT complexis not shown. Khyd represents the rate constantfor the hydrolysisof GTP in the free a* (GTP) species while K[y,~represents the rate constant for the hydrolysisof GTP in any a*(GTP) moleculesthat are still coupled to the photoreeeptor. Koft represents the rate constant for the breakdownof the rhodopsin-a*(GTP)complexwhile Ken representsthe rate constant for the couplingof a(GDP) to rhodopsin.

ment) and GTP hydrolysis (i.e., fluorescence decay). In experiments where relatively low levels of rhodopsin were used (i,e., < 15 nM), the rates of the rhodopsinstimulated binding of GTP were comparable to the rates for GTP hydrolysis. Thus, under these conditions, the extent of the GTP-induced fluorescence enhancement was limited due to the onset of the deactivation of the a T subunit and the accompanying decay of the tzT fluorescence. At higher levels of rhodopsin, the rates for the activation of the total pool of a,r subunits greatly exceeded the rate of GTP hydrolysis, so that under these conditions the fluorescence enhancements directly reflected the GTP binding event. The fluorescence data, obtained at different levels of rhodopsin and GTP, were fit to a model for the rhodops!n-stimulatod GTP binding-GTPase cycle of transducin (Fig. 6) which was developed along the lines of earlier models proposed by Fung [8] and Chabre [7]. In this scheme, light-activated rhodopsin (R) initially couples to the GDP-bound aTT subunit and induces the dis,~aciation of GDP, resulting in a nucleotide-depleted aT subunit. Although as described above, the flY,r subunit complex potentiates the rhodopsin-stimulated

guanine nucleotide exchange within the a T subunit, for simplicity, this species is not shown in the scheme (Fig. 6). It is the rhodopsin-induced dissociation of GDP from the a T subunit which enables the binding of GTP. Ultimately, the aTTGTP species dissociates both from rhodopsin and the fly,r subunit complex. Presumably, it is this dissociation event which accounts for the abilities of both rhodopsin and the /~y,r subunit complex to act catalytically in promoting guanine nucleotide exchange within multiple a,r subunits. It is likely that the bound GTP is hydrolyzed after the aTT subunit has dissociated from rhodopsin and flY'r- This GTPase activity then results in the deactivation of the a T subunit and enables the signaling system to return to its starting point. However, in the scheme shown in Fig. 6, the possibility was considered that at high levels of rhodopsin, a rhodopsin/a-rGTP species may stay intact and hydrolyze GTP at a rate (K[yd) which differs from the rate of hydrolysis of GTP by the free aTT subunit (Khyd). Two assumptions were made to simplify the modeling of the fluorescence data to the schem,z shown in Fig. 6. One of these was that the dissociation of GDP from the rhodopsin-trTT(GDP) complex (K~l) was fast relative to the binding of GTP to the nucleotide-depleted rhodopsin~TT species. This assumption was based on the work of Brandt and Ross which showed that GDP dissociation was not rate-limiP,:~ for the /3-adrenergic receptor-stimulated hindi~!g o f [35S] GTPyS to G~ [30]. The second assumptioL~v~s that the binding of rhodopsin to each molecule of aTT(GDP) (i.e., Kon) was fast relative to the dissociation of a rhodopsin molecule from an activated rhodopsina,r(GTP) complex (i.e., Koff). This assumption was supported by our own experimer.'~l data. Specifically, under conditions where [ a T ] >> [rhod~i~sir~], the activation of the total amount of aT(GDP) ~,,,ould be limited either by the dissociation of rhcdopsin from an activated rhodopsin-a,r(GTP) complex, c,r b~ the subsequent association of the receptor with another molecule of aT(GDP). If the latter step ( K e n ) w e r e rate-limiting, it then would be expected that the rates for the activation of the total pool of a,r would increase as the levels of both rhodopsin and aTT(GDP) were increased. However, this was found not to be the case. Rather, increasing the levels of both rhodopsin and the aTT(GDP) complex (while keeping the ratio of [rhodopsin] to [aT(GDP)] constant) did not change the rate of activation of the aTT subunits. Thus, these results suggested that it is the first-order dissociation of the rhodopsinaT(GTP) complex (Kerr) which limits the rate for the activation of the total a,r(GDP) pool when [a,r(GDP)] >> [rhodopsin]. Fig. 7 shows that the fluorescence data fit well by this simple model with a narrow range of parameters representing the rates for the GTP-binding event

4~ dose-response profiles h~ r h o d o ~ n . ~ m ~ c ~ [~SlGTPyS binding and iy ° ~ | ~ hydro~y.~ ~cd,

A

periments where the [~e~s o~ r~.~J KrcI and ~h~t Kon > Kotr (see text). A: [GTP]= 170 nM. [rhodopsin]= 30 riM. B: [GTP]= 80 nM, [rhodopsin]= 30 riM. C: [GTP] = 42 nM, [rhodopsin] = 30 riM. D: [GTP] = 170 nM, [rhodopsin]= 15 nM. E: [GTP]= 170 nM, [rhodopsin]= 8 riM. These data were taken from Guy et aL [107].

(Kbind = 0.005-0.007/nM

the amoum of r h ~ o ~ m ~m~u~cs i,x~or~r~ed ~ lipid vesicle, were k.kmd~=a|, h~ ~[ c~cs, ~he r h ~ a . ~ n ~-response profiles were c m ~ e n t wilh a ~rn#e bimolecular react/on between r ~ i n and |ransducin and there were no i n d i c a t e s {ha| the ~ i n molecules needed to act cc~3~zradve~ lo e|~i~ ~i~ a~ivat~on of the a~ m ~ c . ~ e s . An important i m p | i c a t ~ derived from the rr~dei. ing of the fhzore~w.er,ce ~ t a was tha~ ~he re,ease of rhodopsin from the r ~ n - a r ( G T P ) s~c~es ma)~ take as long as two seconds within t h ~ e reconstituted phospholipid vesicle s3~ems. This d i ~ c ~ event would occur significant|y faster than GTP ~:,dro~v~, which probably aceoun~ for the finding tha~ r M ~ s i n shows no effect on ~he rate of GTP ~ydro~'ysLs (ke., K~.~ = K ~ ) . Brandt and Ross [36] made a s~[l.ar observation in the case of the $-adrenerg~ r e c e i p t and the GTPase activity of ~he G~ protein. The e~timate for the rate of dissociation of r ~ i n [rom an activated a~(GTP)~ c ~ e s , ob~aine~ f~om ~he fl~mre,~cence studies, was similar lo ( a | t ~ g h s i i ~ t | y km,er than) the estimated rate for th~ d i ~ a t k m event obtained from |ight-scauermg m e ~ a r e m e n t s made in rod outer segments ~ke., ~ S rhodo~sin-av~GTP) complexes were suggested to d i l a t e per s, c.f. Refo ~I~3). These results then raise the p~ssibili~ tha~ the imemetion of an activated a~(GTP) species wi~h the ~ c [ k GMP PDE may cedar prior to the relea~ ~ rhodopsin from the GTP-boand a I subunit.

per s), the dissociation of rhodopsin from the GTP-bound a T subunit (Ko~ = 0.45-1.0/s), and GTP hydrolysis (K~d = K~,.~ = 0.0M0.04/s). In addition, the best-fit parameters for the fluo~e~ence data accurately predicted the rhodopsin

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The dose-response profiles for the effects of the P'tT subunit complex on the r l - ~ and GTPyS-_qim¢lated enhancements of the a T fluorescence have been examined (Figs. 8A, 8B~. Ii was found that /3yT comple~ exerted a strong p r o m o ~ n M effecl on both the GTPTS- and GTP-sdmuiated fluorescence enhancements, consistent with the hnitial work ~ Fang and colleagues, which suggested that the ~TT subanit complex promotes the rhodopsin-stimulated guanine nueleotide exchange reaction within the a r subuni~ [34]. An examination of the rates and the exten~ o~"the rhodopsin- and G T P T S - d e p e ~ n t fluorescence enhancements, as a function of [P~'r] (at cons~am [a-r]~. showed that at least hail- of d~e total a r c c a , a ~ e s could be activated at ratios of t3~0r to a r G D P of ~- 1 : 150 and that greater ~ a n ~ % ~ ~ e to~__] e T G D P molecules were activated within 0.5 rain at a ratio of t ~Y'r to 20 a r molecules.

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~t )",r lpmoil Ftt, I~, The d~pgnder~e of the rhodopsin, and GTP~S-stimulated enhancement of the intrinsic (~'r fluore~ence on/l), T, A: the extent ~,, the rh~=lopstn.~tlmulated enhancement of the a 1, fluorescence as a function of ~YT. Mixtures of the rhodopsin-contulning lipid vesicles (rhodopsl. ! pmol), ¢tTGDP (65 pmot), and/t~/T in the amounts indicated i, the figure were incubated in a total wdume of 14ll pl in room light for 10 mln at 23~ prior to the addition of OTPyS (6'_S nM, final concentration). The ordinate-values represent the relative (rhodopsino and ¢iTPySo~timulated) fluorescence measured at the various levels of $YT, B: plot of the half time of the rhodopsin- and GTP?S-stimulated fluorcgence ~.haneeme~t as a function of/3~,T, The exr~rimental conditions were identical to those described for A. The data for A and B were taken from Ref. 99.

The rates for the GTP),S-stimulated fluorescence ¢nhancetnent, under conditions where [.B'YT] [aTGDP], were: significantly lower than the rates measured when the levels of /3~,T approach those of (ZTGDP (Fig. 8B). One possible explanation for the nonlinear relationship between [~YT] and the rates of (iT activation would be that ¢xT/BYT interactions and/or the coupling of holotransducin to rhodopsin o~ur silnificanily faster than the dissociation of an activated oT suhunit from the ~Y'r subunit complex, If this were the ea~, then st very low levels of/3"~T (i.e., [ ~ T ] '¢ [~X]), the rates tbr the activation of the total

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¢'r pool would be limited by the fact that each new activation event (i.e., GTP~/S binding event) would require the dissociation of /3'~T from the previously activated ¢~TOTPyS species. However, as the levels of ~ ' T approach the levels of ¢zT, the rate for the activation of the total eT pool would no longer be limited by the dissociation of ~8~,T from an aTGTPTS species but rather by the rate of association of each (ZTGDP with OYT, or by the coupling of each holotransducin molecule to rhodopsin. While this type of scheme would yield upward curvatures in plots of the recipro. cal half-time for eT activation versus [/31~T], it does not

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491 fit the actual experimental data well (i.e., the upward curvature in the data shown in Fig. 8B occurs when [/3~,T] is still