Biochimica et B/ophys/ca.4cta, ! 135 (1992) 278-294
287
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4889/92/$05.00
Mmireview
BBAMCR 13193
Recent progress in structure-function and molecular analyses of the pituitary/placental glycoprotein hormone receptors James A. Dias Wad~'orth Center for Laboratories and Researcl~ New York State Department of Health, Albany, N Y (USA)
(Received 26 February 1992)
Key ~rds: Glycoprmein hormone: Hormone receptor;,Pituitaw: Placenta: Signal transduction: Receptor binding; Gene expression; DNA sequence
Contents i.
inrroducdon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
287
!1. Structural aUrt]mtesot the receptors deduced from eDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
!il. Regulation of receptor gene expre~ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
IV. Sm~ure-function studies ~ith receptor mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hormone binding domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . n. CAmtributionof carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Transmembrane and cytoplasmicdomains and signal transduelion . . . . . . . . . . . . . . . . . . . . . V. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rcfe~ces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
290 290 291 292
L Introduction The rece~3tors for the pituitary glycoprotein hormones lu.tropin (LH), thyrotropin (TSH) and follitropin (FSH), and the placental glycoprotein hormone choriogonadotropin (CG) are integral m e m b r a n e proteins, present in vanishing amounts and of remarkable instability. Consequently, they have been studied primarily by virtu,: c,.~ specific interactions with their well defined ligands. The glycoprutein hormones are heterodimeric proteins consisting o f an a a n d p subunit assembled non-covalently [1]. The a subunits of these four glycoprotein hormones are identical in primary structure, but differ in carbohydrate composition [~3]. The ,6 subunits share considerable sequence identity but are
Correspondence: J.A- Dias, Wadswmlh Center for Laboratories and Research, New York State Depa.,Lment of Health, Albany. New York 12201-G509,USA.
292
293 _293
unique, and it has thus been reasoned that hormone specificity at the receptor level is conferred by the /3 subunit. Considerable progress has been m a d e towards understanding the surfaces of the glycoprotein hormone subunits that interact with receptor or with complementary subunit [4], however, a definitive three dimensional structure has not yet been reported. The glycoprotein hormones bind their receptors with similar affinities and all activate the adenylyl cyclase signal transdection pathway and although less well defined, the inositul phosphate pathways [5,6]. The similarity of the glycoprotein hormones and their common pathways of activation of target cells suggested a common structure for their receptors. Until the recent cloning of these receptors which allowed determination of the primary structures, a substantial a m o u n t of information about their biochemical attributes were defined indirectly [7-9]. In 1989, a new understanding about the glycoprotein hormone receptors began when the cloning of the LH
288 and TSH receptor cDNAs was reported. In the ensuing three years the FSH receptor has been cloned and much has be~n learned from analyses of the nucleic acid sequences of these genes, and the application of cDNAs to the study of receptor structure-function and regulation. These studies have had a great impact on our understanding of the mechanism of hormone binding, signal transduction and hormonal control of gene expression for this group of G-protein coupled receptors. in addition they have provided important insights into the pathogenesis of Graves" disease. IL Structural attributes of the receptors deduced from eDNA TSH receptor The human [10-12], rat [13] and canine [14] TSH receptor eDNAs have been cloned and sequenced. At the protein level there is an overall 86-89% homology between TSH receptor from these species [15]. The human TSH receptor eDNA predicted a single polypeptide (plus leader sequence) of 764 amino acids ( M r 86816) [10]. Earlier biochemical studies had indicated that the receptor was composed of subunits and was not a single polypeptide. This issue has been addressed by mutation of TSH receptor cDNA, and expression of a TSH receptor which was resistant to proteolysis. Using this mutant receptor, investigators showed that the two-subunit model for the TSH receptor may reflect proteob~is of the hormone receptor complex during processing. It remains to be determined whethe~ p ~ v n l y t i c processing of the receptor subsequent to hormone binding is necessary and sufficient for signal transduction a n d / o r desensitization. The human TSH receptor ext.racellular domain sequence contains six consensus N-linked glycusylation sites. It is therefore not surpris,~g that the mature protein has an apparent Mr of 100000 by gel electrophoresis [16]. L'I addition, 11 cysteines arc found in the cxtracellular domain and 12 cysteines are found in the transmembrane/cytoplasmic t~omaius. A single consensus protein kinase phosphorylation site is contained within the cytoplasmic domain sequence, Finally, there exists an 8- and a 52-residue insert which is found in the TSH receptor but not the LH or FSH receptors, and this is t~iscnssed in more detail below. FSH receptor The rat [17] and human [18] FSH receptor cDNAs have been cloned and sequenced. There is an overall ho~ology of 89% Uetween the two predicted forms of the protein. The h~mar~ FSH receptor cDNA encodes a single protein oI ¢,78 amino acids ( M r 75465) [17]. There are four potential N-linked glycosylation sites in the human FSH re:eptor amino acid sequence. 11 cysteines are found in tht: extraceilular domain and 12
are found in the mmsmembrane/cytoplasmic domains. Both rat and human FSH receptor C-terminai domains are rich in serine and threonine, which may be potential sites for phosphorylation/regulation. L H receptor The rat [19], porcine [20] and human [21] LH receptor cDNAs have been cloned and sequenced, The human LH receptor is only 42% homologous to the human FSH receptor [18,21]. The human LH receptor shares an 85% homology with the rat LH receptor [21]. The eDNA of the human LH receptor encodes a protein of 674 amino acids ( M r 75632). There are 12 cystcines in the extracellular domain and 13 cysteines in the transmembrane/cytoplasmic domains. The protein sequence has six potential glycosylation sites. Some or all may be glycosylated since the apparent size of iltc LH receptor by gel electrophoresis is M r 93000. Two consensus phusphorylation sites are located in the cytoplasmic domain sequencs. A comparison of the human TSH, LH and FSH receptors reveals the conservation of nine cysteines in their extracellular domain~ and nine cysteines in the transmembrane/cytt~lasmic domains. This suggests that these in particular, are important for folding a n d / o r stab'dization of these domains of all three receptors. All three receptors contain potential sites for phesphorylation within the putative cytoplasmic domain. As a desensitization phenomenon has been observed following tre,?_t~ent of Leydig tumor cells ~i:h ~ [22], these sites are areas of potential interest for further study since phesphorylation of receptors may lead to uncoupling of these receptors following occupancy (desensitization). IIL R e g ~ f i w of receptor g m e The organization of the glycoproteio hormone receptor genes has also proved to be quite interesting. ,The rat LH receptor gene is at least 75 kilobase pmrs and has eleven exons [23-25]. The FSH receptor g e n t spans at least 84 kilobasns of DNA and has 10 exous [26]. The TSH receptor gene spans at least 60 kilobases and consists of 10 exons and nine introus [27]. A feature of the pituitarF glycoprotein hormone receptors, which distinguish them from other G-protein coupled receptors is tttat in addition to their large extracellular domains and large iigands, the extracellular domain is encoded by multiple exons. The exon farthest in the 3" direction in each case e r ~ l e s the putative transmembrane region and the cytoplasmic domain. This is in contrast to other G-protein receptor genes characterized already (for example the gene for the fl-adrenerglc receptor) which have cn~, that exon. I~ such cases the extracellular domains are notably small
289 compared to the extensive large domains of the glycoprotein hormone receptors. Regulation of receptor synthesis by trophic hormones and growth factors is believed to be mediated in part at the level of transcription. The regions of the receptor gene promoters sequenced to date have revealed cyclic AMP response elements, phorbol-ester response eiements and others. A central theme in the field of G-protein coupled receptors is that physiologic levels of trophic hormone lead to a desensitization phenomenon, whi!e pharmacologic levels of trophic hormone lead to a decrease of receptor number. A question, thv,~, was whether this loss in receptor number was preceded by or was a consequence of a decrease in receptor gene expression. TSH receptor
A common theme that emerged from current studies of how receptor levels are regulated is that trophic hormone can cause a transient up-regulation of receptor mRNA, but that prolonged exposure results ip a decrease in receptor mRNA_ T~,e TSH receptor system provides a good example. Physiological levels of TSH decrease m R N A for TSH receptor in as little as 8 h [13]. Reagents that bypass the receptor but activate the cell such as cAMP, forskolin and cholera toxin faithfully replicated this effect. On the other hand phorbol myristate acetate, a non-specific protein kinase stimulator, has no effect on m R N A levels. Similar regulation of the two forms of m R N A (5.6 and 3.3 kilobases) was observed. The decrease in mRlqA is due to an inhibition of initiation of tra.~%:~rip,.~on, not. a ¢h.aqge in h~f-life of the mRNA, and the effect requires serum or insulin-like growth factor-1 (IGFI). A cycioheximide-sensitive protein appears to be no~essary for the down-regulation effect [28]. In contrast, TSH u ~ r e g u latiou of TSH receptor m R N A is not blocked in cycloheximide-treated cells. However, serum or IGFI upregulation of TSH receptor m R N A is blocked by cycloheximide, in summary, trophic hormone can cause a transient increase in receptor mRNA, but eventually what is observed is a loss of receptor mRNA. This hormone action is mediated through the adenylate cyclase system, and a labile protein intermediate appears to be required. L H receptor
In the case of the LH receptor, multiple receptor m R N A transcripts (6.5, 4.5, 2.6, 2.3 and 1.5 kilobases) are coordinately regulated by LH in both testes and ovaries. A decrease in LH receptor m R N A is preceded by a decrease in receptor binding in a testis !.eydig tumor cell line after treatment ,vith hCG [29], In normal Leydig cells, a decrease of all but the smallest receptor m R N A transcripts precedes loss of receptor [30]. It is likely that the difference between normal and
tumor cells, in timing of loss of receptor compared to loss of message is due to ligand induced loss of receptor. For example, bypassing the receptor with either phorbol ester, BtecAMP, or epidermal growth factor results in a decrease of LH receptor m R N A prior to a decrease in LH binding in the Leydig tumor cell line. In the ovary, it has been well documented that folliculogenesis involves an FSH induced increase in LH receptor, and that LH can down-regulate its receptor in luteal cells. The mechanism by which these changes occurred was unknown. The cells in the ovary which contain LH receptor m R N A include granulosa cells and thecal cells [31,32]. It has now been shown that FSH induces LH receptor m R N A in the ovary (preceding an increase in LH binding), and that subsequent treatment with L H / h C G first decreases then increases LH receptor m R N A [30,33-35]. Similar to the testis LH receptor system, LH can up-regulate and down-regulate iI~ own receptor mRNA. However, the timing and exterit of regulation of LH receptor m R N A is clearly influenced by FSH which acts on the same cell, making the regulation of LH receptor m R N A and thus LH receptor, in granulosa cells, different from the Leydig cell system. FSH receptor
FSH receptor m R N A is also present in granulosa cells of the ovary, but whereas treatment with pregnant mare serum gonadotropin (binds FSH receptors) causes an up-regulation of LH receptor m R N A in granulosa cells, FSH receptor m R N A levels in granulosa cells only fluctuate a few fold ~.31,32] Either the FSH receptor gene was insensitive to cAMP-dependent regulation, or alternate mediators .ire involved in the receptor-specific change. Consistent with these in vitro manipulations, and during the course of normal folliculogenesis, LH receptor m R N A increased prior to ovulation and the LH surge, whereas FSH receptors varied only slightly [31,32]. in summary, in the ovary LH receptor m R N A changes appear to be more highly regulate~ than FSH receptor mRNA. In the testis, FSH receptor m R N A is present in Sertoli cells [36,37]. FSH receptor m R N A was decreased by treatment with FSH, and this decrease was not due to a decreased transcriptional initiation [36]. Unlike the TSH receptor system, cycloheximide did not prevent the posttranscriptional decrease of FSH receptor mRNA, saggesting that a labile protein intermediate is not involved in FSH regulation of Sertoli cell FSH receptor mRNA. Testicular FSH receptor m R N A tran.%'ripts include a predominant 2.6 kilobase form and a minor 4.5 kilobase form, and varied threefold (highest at stages 13-2) during the cycle of the seminiferous tubule [37]. The ~tage specificity and continued expression of FSH receptor m R N A in adult rats suggest that FSH plays a role in maintaining sperm output
29O in the adult rat. It remains to be determined if the FSH receptor mRNA regulation by trophic hormone or by Leydig cell androgen, is modulated depending on the stage of the seminiferous epithelium. However, the observation of physiological changes of FSH receptor gone expression in the testis do ~uggest that such regulation is likely. Collectively, these data illustrate that TSH, LH and FSH receptor mRNA is subject to regulation, albeit complex and requiring further study. The ob~.rvations made so far help to explain the regulation of the glycoprotein hormone receptor levels in target organ tissues. 1~: S t r e c t a r e - f u a c f e a studies w i t h receptor mutants
IV-/L Hormone binding domain TSH receptor Structure-functioo studies of glycoprotein hormone receptors have been hampered by the low levels of receptor in tissue. The recent cloning of all three receptors has allowed a comparative, predictive secondary-structure analysis of the different domains. Extensive loop structures and considerable a helix were indicated in all domains [15]. In order to define the bornmne binding domains of these receptors and the
autoimmune antibody-binding domain of the TSH receptor a combination of mutation, synthetic peptide, and chimeric receptor approaches has been used. These are discussed below, and the reader is referred to Fig. 1, for assistance in orientation. TSH receptor mutants that had amino acids 299-301 and 387-395 deleted did not hind TSH implicating these two regions as potential hormone binding sites [38]. Further analysis of this region by site-directed mntagenesis showed that tyrosine-385, threonine-388 and aspartic acid-403 were important for TSH binding [39]. C~teine-301, 390 and 398 were all important for binding and may play a role in formation of a loop which includes the thyroid-stimulating autoantibody epitopo [38,39]. Deletion of 317-366 has no effect on TSH binding or stimulatory autoantibody binding [40]. Residues 303-382 did not confer TSH binding or binding of stimulatory autoantibodies [39]. Within this sequence is the linear epitopo of TSH receptor (352-366) recognized by Graves" disease inhibitory autoantibodies. It is contained within the insert (315-366) unique to TSH, which is bordered by the two sequences important in TSH binding [39,41]. Since Graves' patients autoantibodies bound synthetic peptide 352-366 this region was implicated as an autoimmune epitope [38,41]. Consistent with this line of reasoning was the observation that peptide 333-343 inhibited antibody
..............
,~
Cyt. . . . . . . . . . . . . .
Extrocellulor domain
~
TSHRunique inser(s
Transmembrane domainL_~ Corresponding Exons
2 .... I01
201
301
401
501
O •0
............ dy b;r,dlng sites
e
•
Conserve~ cy~teines
•
a
Possible lycosylation
601
)01
Predicted Amino Acid $eauence ~-'ozitlon
Fig. 1. Strt:ctur¢-functioncorrelates a glycoproteinhormonereceptor. Here, the humanTSH receptor(hTSHR) is emphasizedas an example. The "consen'ed"~steines indicated are not all strictly conserved in the sense identical sequenceposition.The "P' indicates a singlepotential phosphouladonsite. Tlz,ere are two suchsites in hLHR. "Correspondingexons"indicates the regionsthe primau sequencederived from each exen. The boxedareasdo not correlate withexonsize nor are intronsincluded. Other areasand s,~nbolsare defined by the legendsin the figure.
291 binding but did not interact with TSH [42]. A similar peptide (324-344) which includes nine more amino terminal residues has been shown to bind to TSH [43]. Collectively, these data suggest two regions important for TSH binding flank the autoantibody epitope, however, more data are needed to ascertain if the region 324-344 is important. It seems less like!y in view of the observation that deletion of this region had no effect on TSH binding [46]. An additional region that is implicated as a hormone binding site is amino acids 170-260, which confers TSH binding to T S H / L H receptor chimeras [45,46]. Indeed, antipeptide antibodies to either 170202, or to 341-370, have stimulatory activity [47]. Between these two regions, amino acids 201-211 (LH receptor and TSH receptor), and 222-230 (TSH receptor) appear important for binding [48]. An eight amino acid insertion (38-45) unique to TSH receptor appeared important for binding of TSH and autoantibo0ies that stimulate or inhibit TSH receptor [40]. In support of this, a synthetic peptide corresponding to TSH receptor 35-50 blocked binding of TSH to receptor presumably by binding to TSH [49]. Human TSH receptor synthetic peptide corresponding to amino acids 12-30 bound to human TSH [43]. Also, antipeptide antibodies to a region that includes sequence (29-57) had thyroid-stimulating activity but not thyroid inhibitory activity [50], while mutation of cysteine 41 resulted in a decrease in TSH binding [39]. In line with these findings was the observation that a patient with autoimmane hyperthyroidism had a point mutation changing aspartic-36 to histidine [51]. These data collectively indicate that this insertion plays a role in TSH and autoantibody binding. Other autoantibody epitopes in the extracellular loops of the putative transmembrane domains may exist (discussed below). in summary, with regard to the TSHR, the hormone and autoantibody binding domains of the TSH receptor do not seem to be identical sequences. In regard to hormone binding, the early N-terminal and the mid region of the extracellular domain appear important. Some autoantibody epitopes appear near and may overlap the receptor binding regions.
LH and FSH receptors Such detailed structure function studies of the LH and FSH receptor extracellular hormone binding domain have not yet been reported, but the TSH receptor work serves as an example of several successful experimental approaches. In regard to the gonadotropin receptors, truncated cDNA encoding the extracellular domain of the rat LH receptor which does not appear to be membrane bound in this form, binds hCG with high affinity, suggesting that this portion of the receptor is all that is necessary for ligand binding [52,53]. Only one report [52] indicates that the truncated ver-
sions of the receptors are detectable in spent media of transfected cells. At present, ex~racellular forms of receptor are believed to be due to proteolysis. A question that remains is whether any surfaces of the ligand are still exlyoSed following binding to the truncated form of the receptors. If so, then one might predict that additional specificities are conferred by the extracellular domains of the transmembrane regions, which might be important targets for fertility research. In this paradigm, the tigand would be bound by *.he extracellular domain of the receptor, then by some conformational change the ligand or part of the receptor would be brought into contact with an extracellular domain of a transmembrane region, invoking the ensuing activation of adenylate cyclase [53]. Detailed analysis of truncated mutants of the rat LH receptor showed that only the first eight leucine rich motif repeats (amino acids 1-206) are required for high-affinity binding. Chimeric rat FSH receptors that had the N-terminal region replaced with the homologous segment of LH receptor bound hCG [53]. However, the reciprocal experiment (chimeric LH receptor with N-terminal FSH residues) did not bind FSH nor LH. Here, 11 of the 14 |eucine motif repeats were required to confer FSH binding to the LH receptor chimera (amino acids 1-283). It appears by subtractive reasoning, that amino acids within region 140-283 may be important for conferring the FSH-binding phenotype. Species specificity must also be considered when performing these experiments. For example, human LH receptor binds human LH and hCG but not rat LH, whereas rat LH receptor binds hCG as well as rat LH [54]. Therefore an important priority is to reevaluate these structure-function studies with the human re~'~ptors.
IV-B. Contribution of carbohydrate TSH receptor It is possible that nuances in binding specificities as discussed above, may be attributable to differenceg in glycosylation of receptors. The human and rat receptors differ in the number of potential glp:os~'lation sites, and dissimilarities in folding may lead to altered glycosylation states. Between the three human receptors, only two potentml glycosylation sites are conserved. One site conserved at the sequence level is TSHR 198/LHR 195/FSHR 190. The other site conserved in relative position is T S H R 3 0 2 / L H R 294/FSHR 292 (numbering includes leader sequence). Conservation of these sites suggests that they are important to all three receptors for proper folding/and or secretion. However, data collected to date indicate other sites are more importa~lt. Mutation of asparagine-77 or 113 of the human TSH receptor abolished high-affinity TSH binding and cAMP formation,
292 whereas mutation of all other sites had no effect [10]. Whether the decrease in binding activity was due to a loss of receptor at the cell surface was not determined. L H and FSH recet~tors Conversion of one of the conserved glycosylation sites between TSHR aad LHR but not FSHR (asparagine-173; numbering includes leader sequence) to glutamine resulted in a complete loss of cell-surface rat LH receptor;, detergent extraction of cells did not reveal a trapped form of the receptor [55]. Mutation of Asn-291, the other conserved glycos3'lation site, had no effect. Mutation of asparagine-77 and 152 ~esnlted in a reduction of the nambcr of binding sites with no change in affinity [55]. These ~ o important observations underscore the concept that these receptors may have quite different structure-function attributes despite similar sequences. It remains to be determined whether ali sLx potential sites on these receptors are giycosylated. Similar work with follitropin remains to be done, and a comparison between species seems warranted. IV-C. Transmembrane and cytoplasmic donmins and signal transduction TSH, L H and FSH receptors The large extracellular domain of the glycoprotein hormone re--'eptor~ is dhTerent frot~ the short extracellular domain of other G-protein coupled receptors. Analogy with the G-protein coupled receptors with short extracellular domains suggests that part of the effector site resides in the extracellular portions of the transmembrane regions. It was of interest that antipeptide antibodies against the first, second and third loops of the putative transmembrane domain did not block TSH binding to receptor, but antibodies against the second and third loops stimulated cAMP production [56]. In addition, hCG bound with low affinity to truncated rat LH receptors, containing only 10 amino acids of the extracellular domain, and stimulated cAMP production [57]. Such evidence suggests tha~. consideration must be given to a model in which the glycoprotein hormone interacts directly with the extracellular loops of the transmembrane region in addition to a high-affinity interaction with *,he large extracellular domain. The charge of the amino acids within each putative transmembrane segment may also be important for signal transduction since mutation of aspartic acid-383 of the LH receptor to a glutamine resulted in a loss of high affinity binding [58]. The phenomenon of desensitization may involve the cytoplasmic tail of the receptor. Treatment of Leydig tumor cells with antisense oligonucleotides that code for the third extracellular loop of the rat LH receptor produced truncated receptor and prevented desensitization induced by LH, PMA or BtecAMP [22]. The'=e
results suggest that the intracellular cytoplasmic tail of the LH receptor is required for the desensitization process to occur, possibly through phosphorylation of sites on the C-terminal cytoplasmic end of the receptor. The three cytoplasmic loops and the C-terminal tail of the receptors are potential sites of interactions with the G-psoteins. Mutations in the first cytoplasmic loop and the carboxyl ends of the second and third cytoplasmic loops of TSH receptor cause a loss of signal transduction [59]. The Coterminal region 709-764 is not needed for "ISH action. Mutations in the amino terminus of the second loop, and in the C-terminal end of the third loop, resulted in a decrease in affinity of ligand binding which made it difficult to interpret a decrease in signal transduction. Attempts to further define specific amino acids in the C-terminal end of the third cytoplasmic loop revealed that no single residue was important for signal transductlon [60]. These data support the concept that the cytoplasmic domains of the receptor are involved in signal transduction. V. Futare d i r ~ The cloning of the cDNAs for the glycoprotein receptors has allowed deduction of protein sequence information from which reasonable hypotheses about siru~i,~re and function could be formulated. Elucidation of the primary structures of the glycoprotein hormones can be used as an example of how such information led to important structure and function correlates. However, in the case of the hormones, as will be the case with the receptors, the information gained by such studies can only pinpoint the sequences, and specific amino acids, involved in functional attributes. It should be clear from the foregoing discussion that hormone binding by the receptor involves discontinuous sequences. Whether these sequences are assembled, to form a discontinuous epitope will require ~ r t b e r tertiary analysis. One approach would use monoclonal antibodies prepared by immunizing with the receptors. These antibodies can be epitope mapped with synthetic or recomb',ant receptor peptides in order to determine the dis~ontinuous sequences which form regional su~aces on the receptors. It may be that the binding determinants of both the giycoprotein hormones and their receptors which c6nfor 'specificity', depend on conformatioaal diversity (in ~ e face of sequence conservation). If so, the three-dimensional structures of each hormone and receptor will be required to develop a better understanding of this interaction. Of course, since it is quite possible that c~nformatiou~l changes may occur as a result of the binding interaction, the three dimensional structure of the complex as well as the individual proteins will have to be determined. It would be desirable to
293 formulate h y p o t h e s e s b a s e d o n a t h r e e dimensional structure r a t h e r t h a n trying to a p p r o x i m a t e c o n f o r m a tional attributes. T h e t h r e e d i m e n s i o n a l s t r u c t u r e o f the g r o w t h h o r m o n e - r e c e p t o r complex [61] h a s b e e n d e t e r m i n e d . Suitable high level expression o f insulin r e c e p t o r [62], L H r e c e p t o r [15] a n d o t h e r G - p r o t e i n c o u p l e d r e c e p t o r s [63] in insect cells h a s b e e n achieved. T h u s , t h e p r o d u c t i o n o f sufficient p r o t e i n to p e r f o r m crystal!ixation trials a n d ultimately to allow crystallization o f t h e reclzptors s h o u l d b e a n a r e a o f high priority for investigators o f t h e glycoprotein h o r m o n e r e c e p t o r s a n d t h e i r ligands. T h e level o f r e c e p t o r p r o t e i n o n the cell s u r f a c e a p p e a r s to be subject t o r e g u l a t i o n a t several levels, including g e n ¢ expression, a n d possibly proteolysis a n d p h o s p h o w l a t i o n . F u t u r e investigations will b e challenging since e a c h o f these p a r t i c u l a r m e c h a n i s m s for regulatinu im, olves incompletely u n d e r s t o o d hierarchical complex r e g u l a t o r y c o m p o n e n t s . Yet n e w a n d i m p o r t a n t i n f o r m a t i o n h a s a l r e a d y b e e n g a i n e d f r o m early w o r k in this a r e a . Analysis o f p r o m o t e r regions u p s t r e a m o f t r a n s c r i p tional s t a r t sites suggests t h a t up- a n d d o w n - r e g u l a t i o n o f t h e glycuprotein h o r m o n e r e c e p t o r g e n e s m a y involve b o t h c A M P - d e p e n d e n t a n d - i n d e p e n d e n t m e c h a nisms. Certainly, t h e c A M P down*regulation o f r e c e p t o r g e n e expression is a n interesting a r e a o f study. M u c h p r o t e i n chemistry will n e e d t o b e d o n e to identify w h e t h e r p h u s p h o r y l a t i o n o f e a c h o f t h e t h r e e glycoprotein h o r m o n e r e c e p t o r s o c c u r s a n d w h e t h e r desensitization is a c o m m o n p a t h w a y t o a t t e n u a t i o n o f cellular response. W h e t h e r o r n o t all potential glycosylation sites h a v e c a r b o h y d r a t e , a n d t h e c a r b o h y d r a t e s t r u c t u r e s p r e s e n t , a s well a s the diversity o f glycosylation a n d complexity b e t w e e n t h e receptors, as well as b e t w e e n species, will also n e e d t o b e d e t e r m i n e d . T h e clinical a n d p u b l i c h e a l t h i m p a c t o f w o r k to d a t e a n d f u t u r e w o r k is likely to b e great. A n obvious e x a m p l e is t h e identification o f the d e t e r m i n a n t s o f a u t o i m m u n i t y in Graves" disease. It c a n b e e x p e c t e d t h a t n e w u n d e r s t a n d i n g s o f diseases o f thyroid function a n d o f infertility will b e f o r t h c o m i n g . In addition, n e w a p p r o a c h e s to fertility c o n t r o l m a y also result. Acknowledgement T h i s study w a s s u p p o r t e d b y N I H H D 1 8 4 0 7 . References I pierce, J. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50. 465-495. 2 Baenziger, J.U. and Green, E.D. (1988) Biochim. Biophys. Acta 947, 287-306. 3 Green, E.D. and Baenziger, J.U. (19881 J. Biol. Chem. 263. 25-35. 4 Dias, J.A. (1992.)Trends EndocrinoL Metabo 3, 24-29.
5 Philip, N.J. and Grollamn, E.F. (19861 Febs Le~t. 202, 193-196. 6 Gudermann, T., Birnbaumer, M. and Bimbaumer, L (1922) J. Biol. Chem. 267, 4479-4488. 7 Ascoli, M. and Segaloff, D.L. (19891Endocr. Re,/. 10, 27-44. 8 Rees Smith, B. and Furmaniak, L (19901 Horm. Metab. Res. Suppl. 23, 28-32. 9 Rees Smith, R., McLachian, S.M. and Furmaniak, J. (19881 Endocr. Rev. 9,106-121. 10 Nagayama, Y., Kaufman, K.D., Seto, P. and RapoporL B. (19891 Biochem. Biophys. Res. Commun. 165,1184-1190. I I Misrahi, M., Loosfelt, H., Atger, M, Sar, S., Guiochon-Mantel, A. and Milgrom, E. (19901Biochem. Bloph~. Res. Commun. 166, 394-403. 12 Libert, F., Lefort, A., Gerard, C., et al. (1989) Biochem. Biophys. Res. Commun. 165, 1250-1255. 13 Akamizu, T., Ikuyama, S., Saji, M., et aL (1990) Proc. Natl. Acad. of Sci. USA 87, 5677-5681. 14 Parmentier, M., Libert, F., Maenhaut, C., el al. (1989) Science 246, 1620-1622. 15 Salesse, R., Remy, J.J., Levin, J.M., Jallal, B. and Gamier, J. (19911 Biochimie 73, 109-120. 16 Russo, D., Chazcnbalk, G.D., Nagayama, Y., Wadsworth, H.L. and Rapopost B. (19911Mol. Endocrinol. 5, 29-33. 17 Sprengel, R., Braun, T., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (19901 Mnl. EndocrinoL 4, 525-530. 18 Minegish T, Nakamura, K., Takakura, Y., Ibuki, Y. and lgarashi. M. (19911 Biochem. Biophys, Res. Commun. 175, !125-1130. 19 McFarland KC., Sprengel, R., Phillips, H.S., et aL (19891Science 245, 494-499. 20 Loosfelt, H.. Mist3hi, M., Alger, M., et al. (19891 Science 245, 525-528. 21 Minegish, T., Nakamura, K., Takakura, Y., et al. (19901Biochem. Bioph~. Res. Commun. 172,1049-1054. 22 West, A.P. and Cooke, B.A. (19911 Mol. Cell. Endoc.!r,ol. 79, Rg-RI4. 23 Tsai-Morris, C.H., Buczko, E., Wang, W. and Dufau, M.L (19901 J. Biol. Chem. 265,19385-19388. 24 Tsai-Morris, C.H., Buczko, E., Wang, W., Xie, X.Z. and Dufau, M,L. (1991)J. Biol. Chem. 266,11355-11359. 25 Koo, Y.B., Ji, l., Slaughter, R.G. and Ji, T.H. (10911Endo,.minolot~ 128, 2297-23o8. 26 He':kert, Lid., Dalcy, IJ. and Griswold, M.D. (1992) Mol. Endocrino!. 6, 70-80. 27 Gross, B., Misrahi, M., Sar, S. and Milgrom, E. (19911Biochem. Biophys. Res. Commun. 177, 679-687. 28 Saji, M., Akamizu, T., Sanchez, M., et aL 0992) Endocrinology 130,'520-533. 29 Wang, H., Segaloff, D.L and Ascoli, M. (19911 J. Biol. Chem. 266, 780-785. 30 LaPolt, ES., Jia, X.C., Sincich, C. and Hsueh, AJ. (1991) Mol. Endocrinol. 5, 397-403. 31 Camp, T.A., Rahal, J.O. and Mayo, K.E. (19911Mol. Endocrinol. 5, 1405-1417. 32 Peng, X., Hsueh, AJ.W., LaPolt, P.S., Bjersing, L. and Ny, T. (19911 Endocrinology 129, 3200-3207. 33 Hoffman, Y.M., Peegel, H., Sprock, MJ., Zhang, O-Y- and Menon, K.M. (19911Endocrinology 12,3,388-393. 34 LaPolt, P.S., Oikawa, M., Jia, X.C., Dargan, C. and Hsueh, AJ. (19901 Endocrinology 126, 3277-3279. 35 Nakamura, K., Minegishi, T., Takakura, Y., et al. (19901Biochem. Biophys. Res. Commun. 172, 786-792. 36 Themmen, A.P.N., Blok, LJ., Post, M., et al. (19911 Mol. Ceil. EndocrinoL 78, R.7-RA3. 37 Heckert, L.L. and Griswold, M.D. (;~"91) Mol. Endocrinol. 5, 670-677. 38 Kosugi, S., Ban, T., Akamizu, t. and Kohn, LD. (19911J. Biol. Chem. 266. 19413-19418.
294 39 Kosugi, S . Ban, T . Akamizu, T. and Kohn, L D . (1991) Biochem. Biophys. Res. Commun. 180, i!i8-1124. 40 Wadsworth, H.L. Chazenbalk, G . D . Nagayama. Y . Russo, D. and Rapoport 13. (1990) Science 249,1423-1425. 41 TakaL O . Desai, ILK., Seetharamaiah, G.S. et aL (1991) Biochem. Bioph~ Res. Commun. 179, 319-326. 42 Moil, T . Sugawa, l-L, Piraphatdist, T . lnoue, D . Enomoto, T. and lmura, H. (1991) Biochem. Biophys. Res. Commun. 178, 165-172. 43 Atassl. M.Z., Manshougi, T. and Sakata, S. (1991) Proc. Natl. Acad. Sci. USA 88, 3613-3617. 44 West, A.P. and Cooke, B.A. (1991) Endocrinology 128, 363-370. 45 Nagayama. Y . Russo, 13. Chazanbalk, G.D. Wadsworth, H.L. and Rapoport B. {1990) Biochem. Biophys. Res. Commun. 173, 1150-1156. 46 Nagayama, Y . Wadsworth, I L L . Chazenbalk, G . D . Russo, D . Seto. P. and Rapoport, 13. (1991) Proc. Natl. Acad. Sci. USA 88, 902-905. 47 Endo, T . Ohmori, M . lkeda, M . Kotani, S. and Onaya, T. (1991) Biochem. Biophys. Res. Commun. 179,1548-1553. 48 Nagayama. Y . Russo, D . Wadsworth, H.L, ChazenbalL G.D. and Rapoport B. (1991)£ Biol. Chem. 266.14926-14930. 4¢) Ohmori, M . Endo, T . lknda, M. and Onaya, T. (1991) Biochem. Biopl~'s. Res. Connnun. 178, 733-738. 50 Endo, T . Ohmor/. M . lkeda, M. and Onaya, T. (1991) Biochem. Bioph~. R¢~ Cornman. IT/. 145-150.
51 H c l d ~ N . Gustavsson, B . Westermark. IC and Westermark, B. (1991) J. Clio. EndocriooL Metab. 73, 1374-1376. 52 Xi¢, Y.B., Wang. H. and Scgaloff, D.L. (1990) J. Biol. Chem. 265, 21411-21414. 53 I[haun, T~ Schofmld, PAL and Spreng¢l, IL (1991) EMBO J. 10. 1885-1890. 54 Jia, X.C. ~ M . 13o, M . et aL (1990 Mol. Endocrinol. 5, 759-768. 55 Zlmng, R., Tsai Morris, C.H., Kitamura, M., Buczko, E. and Dufau, M. (1991) Biochem. Biophys. Res. Commun. 181, 808. 56 Endo, T~ Ohmori, M., lkeda, M. and Onaya, T. (1991) Biochem. Biophys. Res. Commun. 181, 10_~-!04t. 57 JL I.EL and Ji, T.IL (1991~"L Biol. Chem. 266,13076-13079. 58 Ji, !. and Ji, T.H. (1991) J. Biol. Chem. 266,14953-14957. 59 Chazenbalk, G . D . Nagayama. Y., Russo, D . Wadsworth, H.L. and Rapolam B. (1990) J. Biol. Chem. 265. 20970-20975. 60 Chazenbalk. I L D . Nagayama. Y . Wadsworth, H . Russo, D. and Rapopo~ B. (1991) MoL Endocrinol. 5, i523-1526. 61 Devos, A M . Ultsch, M. and Kossiakoff, A.A. (1992) Science 255, 3116-312. 62 Paul, J . L Tavareo L Denton, ILM. and Steioer, D.F. (1990) J. Biol. Chem.. 265,13074-13083. 63 Parker, E.M.. Kameyam& K., Higashijima. T. and Ross, E.M. (1991) J. BioL Chem. 266, 519-527.
287
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4889/92/$05.00
Mmireview
BBAMCR 13193
Recent progress in structure-function and molecular analyses of the pituitary/placental glycoprotein hormone receptors James A. Dias Wad~'orth Center for Laboratories and Researcl~ New York State Department of Health, Albany, N Y (USA)
(Received 26 February 1992)
Key ~rds: Glycoprmein hormone: Hormone receptor;,Pituitaw: Placenta: Signal transduction: Receptor binding; Gene expression; DNA sequence
Contents i.
inrroducdon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
287
!1. Structural aUrt]mtesot the receptors deduced from eDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
!il. Regulation of receptor gene expre~ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
IV. Sm~ure-function studies ~ith receptor mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hormone binding domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . n. CAmtributionof carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Transmembrane and cytoplasmicdomains and signal transduelion . . . . . . . . . . . . . . . . . . . . . V. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rcfe~ces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
290 290 291 292
L Introduction The rece~3tors for the pituitary glycoprotein hormones lu.tropin (LH), thyrotropin (TSH) and follitropin (FSH), and the placental glycoprotein hormone choriogonadotropin (CG) are integral m e m b r a n e proteins, present in vanishing amounts and of remarkable instability. Consequently, they have been studied primarily by virtu,: c,.~ specific interactions with their well defined ligands. The glycoprutein hormones are heterodimeric proteins consisting o f an a a n d p subunit assembled non-covalently [1]. The a subunits of these four glycoprotein hormones are identical in primary structure, but differ in carbohydrate composition [~3]. The ,6 subunits share considerable sequence identity but are
Correspondence: J.A- Dias, Wadswmlh Center for Laboratories and Research, New York State Depa.,Lment of Health, Albany. New York 12201-G509,USA.
292
293 _293
unique, and it has thus been reasoned that hormone specificity at the receptor level is conferred by the /3 subunit. Considerable progress has been m a d e towards understanding the surfaces of the glycoprotein hormone subunits that interact with receptor or with complementary subunit [4], however, a definitive three dimensional structure has not yet been reported. The glycoprotein hormones bind their receptors with similar affinities and all activate the adenylyl cyclase signal transdection pathway and although less well defined, the inositul phosphate pathways [5,6]. The similarity of the glycoprotein hormones and their common pathways of activation of target cells suggested a common structure for their receptors. Until the recent cloning of these receptors which allowed determination of the primary structures, a substantial a m o u n t of information about their biochemical attributes were defined indirectly [7-9]. In 1989, a new understanding about the glycoprotein hormone receptors began when the cloning of the LH
288 and TSH receptor cDNAs was reported. In the ensuing three years the FSH receptor has been cloned and much has be~n learned from analyses of the nucleic acid sequences of these genes, and the application of cDNAs to the study of receptor structure-function and regulation. These studies have had a great impact on our understanding of the mechanism of hormone binding, signal transduction and hormonal control of gene expression for this group of G-protein coupled receptors. in addition they have provided important insights into the pathogenesis of Graves" disease. IL Structural attributes of the receptors deduced from eDNA TSH receptor The human [10-12], rat [13] and canine [14] TSH receptor eDNAs have been cloned and sequenced. At the protein level there is an overall 86-89% homology between TSH receptor from these species [15]. The human TSH receptor eDNA predicted a single polypeptide (plus leader sequence) of 764 amino acids ( M r 86816) [10]. Earlier biochemical studies had indicated that the receptor was composed of subunits and was not a single polypeptide. This issue has been addressed by mutation of TSH receptor cDNA, and expression of a TSH receptor which was resistant to proteolysis. Using this mutant receptor, investigators showed that the two-subunit model for the TSH receptor may reflect proteob~is of the hormone receptor complex during processing. It remains to be determined whethe~ p ~ v n l y t i c processing of the receptor subsequent to hormone binding is necessary and sufficient for signal transduction a n d / o r desensitization. The human TSH receptor ext.racellular domain sequence contains six consensus N-linked glycusylation sites. It is therefore not surpris,~g that the mature protein has an apparent Mr of 100000 by gel electrophoresis [16]. L'I addition, 11 cysteines arc found in the cxtracellular domain and 12 cysteines are found in the transmembrane/cytoplasmic t~omaius. A single consensus protein kinase phosphorylation site is contained within the cytoplasmic domain sequence, Finally, there exists an 8- and a 52-residue insert which is found in the TSH receptor but not the LH or FSH receptors, and this is t~iscnssed in more detail below. FSH receptor The rat [17] and human [18] FSH receptor cDNAs have been cloned and sequenced. There is an overall ho~ology of 89% Uetween the two predicted forms of the protein. The h~mar~ FSH receptor cDNA encodes a single protein oI ¢,78 amino acids ( M r 75465) [17]. There are four potential N-linked glycosylation sites in the human FSH re:eptor amino acid sequence. 11 cysteines are found in tht: extraceilular domain and 12
are found in the mmsmembrane/cytoplasmic domains. Both rat and human FSH receptor C-terminai domains are rich in serine and threonine, which may be potential sites for phosphorylation/regulation. L H receptor The rat [19], porcine [20] and human [21] LH receptor cDNAs have been cloned and sequenced, The human LH receptor is only 42% homologous to the human FSH receptor [18,21]. The human LH receptor shares an 85% homology with the rat LH receptor [21]. The eDNA of the human LH receptor encodes a protein of 674 amino acids ( M r 75632). There are 12 cystcines in the extracellular domain and 13 cysteines in the transmembrane/cytoplasmic domains. The protein sequence has six potential glycosylation sites. Some or all may be glycosylated since the apparent size of iltc LH receptor by gel electrophoresis is M r 93000. Two consensus phusphorylation sites are located in the cytoplasmic domain sequencs. A comparison of the human TSH, LH and FSH receptors reveals the conservation of nine cysteines in their extracellular domain~ and nine cysteines in the transmembrane/cytt~lasmic domains. This suggests that these in particular, are important for folding a n d / o r stab'dization of these domains of all three receptors. All three receptors contain potential sites for phesphorylation within the putative cytoplasmic domain. As a desensitization phenomenon has been observed following tre,?_t~ent of Leydig tumor cells ~i:h ~ [22], these sites are areas of potential interest for further study since phesphorylation of receptors may lead to uncoupling of these receptors following occupancy (desensitization). IIL R e g ~ f i w of receptor g m e The organization of the glycoproteio hormone receptor genes has also proved to be quite interesting. ,The rat LH receptor gene is at least 75 kilobase pmrs and has eleven exons [23-25]. The FSH receptor g e n t spans at least 84 kilobasns of DNA and has 10 exous [26]. The TSH receptor gene spans at least 60 kilobases and consists of 10 exons and nine introus [27]. A feature of the pituitarF glycoprotein hormone receptors, which distinguish them from other G-protein coupled receptors is tttat in addition to their large extracellular domains and large iigands, the extracellular domain is encoded by multiple exons. The exon farthest in the 3" direction in each case e r ~ l e s the putative transmembrane region and the cytoplasmic domain. This is in contrast to other G-protein receptor genes characterized already (for example the gene for the fl-adrenerglc receptor) which have cn~, that exon. I~ such cases the extracellular domains are notably small
289 compared to the extensive large domains of the glycoprotein hormone receptors. Regulation of receptor synthesis by trophic hormones and growth factors is believed to be mediated in part at the level of transcription. The regions of the receptor gene promoters sequenced to date have revealed cyclic AMP response elements, phorbol-ester response eiements and others. A central theme in the field of G-protein coupled receptors is that physiologic levels of trophic hormone lead to a desensitization phenomenon, whi!e pharmacologic levels of trophic hormone lead to a decrease of receptor number. A question, thv,~, was whether this loss in receptor number was preceded by or was a consequence of a decrease in receptor gene expression. TSH receptor
A common theme that emerged from current studies of how receptor levels are regulated is that trophic hormone can cause a transient up-regulation of receptor mRNA, but that prolonged exposure results ip a decrease in receptor mRNA_ T~,e TSH receptor system provides a good example. Physiological levels of TSH decrease m R N A for TSH receptor in as little as 8 h [13]. Reagents that bypass the receptor but activate the cell such as cAMP, forskolin and cholera toxin faithfully replicated this effect. On the other hand phorbol myristate acetate, a non-specific protein kinase stimulator, has no effect on m R N A levels. Similar regulation of the two forms of m R N A (5.6 and 3.3 kilobases) was observed. The decrease in mRlqA is due to an inhibition of initiation of tra.~%:~rip,.~on, not. a ¢h.aqge in h~f-life of the mRNA, and the effect requires serum or insulin-like growth factor-1 (IGFI). A cycioheximide-sensitive protein appears to be no~essary for the down-regulation effect [28]. In contrast, TSH u ~ r e g u latiou of TSH receptor m R N A is not blocked in cycloheximide-treated cells. However, serum or IGFI upregulation of TSH receptor m R N A is blocked by cycloheximide, in summary, trophic hormone can cause a transient increase in receptor mRNA, but eventually what is observed is a loss of receptor mRNA. This hormone action is mediated through the adenylate cyclase system, and a labile protein intermediate appears to be required. L H receptor
In the case of the LH receptor, multiple receptor m R N A transcripts (6.5, 4.5, 2.6, 2.3 and 1.5 kilobases) are coordinately regulated by LH in both testes and ovaries. A decrease in LH receptor m R N A is preceded by a decrease in receptor binding in a testis !.eydig tumor cell line after treatment ,vith hCG [29], In normal Leydig cells, a decrease of all but the smallest receptor m R N A transcripts precedes loss of receptor [30]. It is likely that the difference between normal and
tumor cells, in timing of loss of receptor compared to loss of message is due to ligand induced loss of receptor. For example, bypassing the receptor with either phorbol ester, BtecAMP, or epidermal growth factor results in a decrease of LH receptor m R N A prior to a decrease in LH binding in the Leydig tumor cell line. In the ovary, it has been well documented that folliculogenesis involves an FSH induced increase in LH receptor, and that LH can down-regulate its receptor in luteal cells. The mechanism by which these changes occurred was unknown. The cells in the ovary which contain LH receptor m R N A include granulosa cells and thecal cells [31,32]. It has now been shown that FSH induces LH receptor m R N A in the ovary (preceding an increase in LH binding), and that subsequent treatment with L H / h C G first decreases then increases LH receptor m R N A [30,33-35]. Similar to the testis LH receptor system, LH can up-regulate and down-regulate iI~ own receptor mRNA. However, the timing and exterit of regulation of LH receptor m R N A is clearly influenced by FSH which acts on the same cell, making the regulation of LH receptor m R N A and thus LH receptor, in granulosa cells, different from the Leydig cell system. FSH receptor
FSH receptor m R N A is also present in granulosa cells of the ovary, but whereas treatment with pregnant mare serum gonadotropin (binds FSH receptors) causes an up-regulation of LH receptor m R N A in granulosa cells, FSH receptor m R N A levels in granulosa cells only fluctuate a few fold ~.31,32] Either the FSH receptor gene was insensitive to cAMP-dependent regulation, or alternate mediators .ire involved in the receptor-specific change. Consistent with these in vitro manipulations, and during the course of normal folliculogenesis, LH receptor m R N A increased prior to ovulation and the LH surge, whereas FSH receptors varied only slightly [31,32]. in summary, in the ovary LH receptor m R N A changes appear to be more highly regulate~ than FSH receptor mRNA. In the testis, FSH receptor m R N A is present in Sertoli cells [36,37]. FSH receptor m R N A was decreased by treatment with FSH, and this decrease was not due to a decreased transcriptional initiation [36]. Unlike the TSH receptor system, cycloheximide did not prevent the posttranscriptional decrease of FSH receptor mRNA, saggesting that a labile protein intermediate is not involved in FSH regulation of Sertoli cell FSH receptor mRNA. Testicular FSH receptor m R N A tran.%'ripts include a predominant 2.6 kilobase form and a minor 4.5 kilobase form, and varied threefold (highest at stages 13-2) during the cycle of the seminiferous tubule [37]. The ~tage specificity and continued expression of FSH receptor m R N A in adult rats suggest that FSH plays a role in maintaining sperm output
29O in the adult rat. It remains to be determined if the FSH receptor mRNA regulation by trophic hormone or by Leydig cell androgen, is modulated depending on the stage of the seminiferous epithelium. However, the observation of physiological changes of FSH receptor gone expression in the testis do ~uggest that such regulation is likely. Collectively, these data illustrate that TSH, LH and FSH receptor mRNA is subject to regulation, albeit complex and requiring further study. The ob~.rvations made so far help to explain the regulation of the glycoprotein hormone receptor levels in target organ tissues. 1~: S t r e c t a r e - f u a c f e a studies w i t h receptor mutants
IV-/L Hormone binding domain TSH receptor Structure-functioo studies of glycoprotein hormone receptors have been hampered by the low levels of receptor in tissue. The recent cloning of all three receptors has allowed a comparative, predictive secondary-structure analysis of the different domains. Extensive loop structures and considerable a helix were indicated in all domains [15]. In order to define the bornmne binding domains of these receptors and the
autoimmune antibody-binding domain of the TSH receptor a combination of mutation, synthetic peptide, and chimeric receptor approaches has been used. These are discussed below, and the reader is referred to Fig. 1, for assistance in orientation. TSH receptor mutants that had amino acids 299-301 and 387-395 deleted did not hind TSH implicating these two regions as potential hormone binding sites [38]. Further analysis of this region by site-directed mntagenesis showed that tyrosine-385, threonine-388 and aspartic acid-403 were important for TSH binding [39]. C~teine-301, 390 and 398 were all important for binding and may play a role in formation of a loop which includes the thyroid-stimulating autoantibody epitopo [38,39]. Deletion of 317-366 has no effect on TSH binding or stimulatory autoantibody binding [40]. Residues 303-382 did not confer TSH binding or binding of stimulatory autoantibodies [39]. Within this sequence is the linear epitopo of TSH receptor (352-366) recognized by Graves" disease inhibitory autoantibodies. It is contained within the insert (315-366) unique to TSH, which is bordered by the two sequences important in TSH binding [39,41]. Since Graves' patients autoantibodies bound synthetic peptide 352-366 this region was implicated as an autoimmune epitope [38,41]. Consistent with this line of reasoning was the observation that peptide 333-343 inhibited antibody
..............
,~
Cyt. . . . . . . . . . . . . .
Extrocellulor domain
~
TSHRunique inser(s
Transmembrane domainL_~ Corresponding Exons
2 .... I01
201
301
401
501
O •0
............ dy b;r,dlng sites
e
•
Conserve~ cy~teines
•
a
Possible lycosylation
601
)01
Predicted Amino Acid $eauence ~-'ozitlon
Fig. 1. Strt:ctur¢-functioncorrelates a glycoproteinhormonereceptor. Here, the humanTSH receptor(hTSHR) is emphasizedas an example. The "consen'ed"~steines indicated are not all strictly conserved in the sense identical sequenceposition.The "P' indicates a singlepotential phosphouladonsite. Tlz,ere are two suchsites in hLHR. "Correspondingexons"indicates the regionsthe primau sequencederived from each exen. The boxedareasdo not correlate withexonsize nor are intronsincluded. Other areasand s,~nbolsare defined by the legendsin the figure.
291 binding but did not interact with TSH [42]. A similar peptide (324-344) which includes nine more amino terminal residues has been shown to bind to TSH [43]. Collectively, these data suggest two regions important for TSH binding flank the autoantibody epitope, however, more data are needed to ascertain if the region 324-344 is important. It seems less like!y in view of the observation that deletion of this region had no effect on TSH binding [46]. An additional region that is implicated as a hormone binding site is amino acids 170-260, which confers TSH binding to T S H / L H receptor chimeras [45,46]. Indeed, antipeptide antibodies to either 170202, or to 341-370, have stimulatory activity [47]. Between these two regions, amino acids 201-211 (LH receptor and TSH receptor), and 222-230 (TSH receptor) appear important for binding [48]. An eight amino acid insertion (38-45) unique to TSH receptor appeared important for binding of TSH and autoantibo0ies that stimulate or inhibit TSH receptor [40]. In support of this, a synthetic peptide corresponding to TSH receptor 35-50 blocked binding of TSH to receptor presumably by binding to TSH [49]. Human TSH receptor synthetic peptide corresponding to amino acids 12-30 bound to human TSH [43]. Also, antipeptide antibodies to a region that includes sequence (29-57) had thyroid-stimulating activity but not thyroid inhibitory activity [50], while mutation of cysteine 41 resulted in a decrease in TSH binding [39]. In line with these findings was the observation that a patient with autoimmane hyperthyroidism had a point mutation changing aspartic-36 to histidine [51]. These data collectively indicate that this insertion plays a role in TSH and autoantibody binding. Other autoantibody epitopes in the extracellular loops of the putative transmembrane domains may exist (discussed below). in summary, with regard to the TSHR, the hormone and autoantibody binding domains of the TSH receptor do not seem to be identical sequences. In regard to hormone binding, the early N-terminal and the mid region of the extracellular domain appear important. Some autoantibody epitopes appear near and may overlap the receptor binding regions.
LH and FSH receptors Such detailed structure function studies of the LH and FSH receptor extracellular hormone binding domain have not yet been reported, but the TSH receptor work serves as an example of several successful experimental approaches. In regard to the gonadotropin receptors, truncated cDNA encoding the extracellular domain of the rat LH receptor which does not appear to be membrane bound in this form, binds hCG with high affinity, suggesting that this portion of the receptor is all that is necessary for ligand binding [52,53]. Only one report [52] indicates that the truncated ver-
sions of the receptors are detectable in spent media of transfected cells. At present, ex~racellular forms of receptor are believed to be due to proteolysis. A question that remains is whether any surfaces of the ligand are still exlyoSed following binding to the truncated form of the receptors. If so, then one might predict that additional specificities are conferred by the extracellular domains of the transmembrane regions, which might be important targets for fertility research. In this paradigm, the tigand would be bound by *.he extracellular domain of the receptor, then by some conformational change the ligand or part of the receptor would be brought into contact with an extracellular domain of a transmembrane region, invoking the ensuing activation of adenylate cyclase [53]. Detailed analysis of truncated mutants of the rat LH receptor showed that only the first eight leucine rich motif repeats (amino acids 1-206) are required for high-affinity binding. Chimeric rat FSH receptors that had the N-terminal region replaced with the homologous segment of LH receptor bound hCG [53]. However, the reciprocal experiment (chimeric LH receptor with N-terminal FSH residues) did not bind FSH nor LH. Here, 11 of the 14 |eucine motif repeats were required to confer FSH binding to the LH receptor chimera (amino acids 1-283). It appears by subtractive reasoning, that amino acids within region 140-283 may be important for conferring the FSH-binding phenotype. Species specificity must also be considered when performing these experiments. For example, human LH receptor binds human LH and hCG but not rat LH, whereas rat LH receptor binds hCG as well as rat LH [54]. Therefore an important priority is to reevaluate these structure-function studies with the human re~'~ptors.
IV-B. Contribution of carbohydrate TSH receptor It is possible that nuances in binding specificities as discussed above, may be attributable to differenceg in glycosylation of receptors. The human and rat receptors differ in the number of potential glp:os~'lation sites, and dissimilarities in folding may lead to altered glycosylation states. Between the three human receptors, only two potentml glycosylation sites are conserved. One site conserved at the sequence level is TSHR 198/LHR 195/FSHR 190. The other site conserved in relative position is T S H R 3 0 2 / L H R 294/FSHR 292 (numbering includes leader sequence). Conservation of these sites suggests that they are important to all three receptors for proper folding/and or secretion. However, data collected to date indicate other sites are more importa~lt. Mutation of asparagine-77 or 113 of the human TSH receptor abolished high-affinity TSH binding and cAMP formation,
292 whereas mutation of all other sites had no effect [10]. Whether the decrease in binding activity was due to a loss of receptor at the cell surface was not determined. L H and FSH recet~tors Conversion of one of the conserved glycosylation sites between TSHR aad LHR but not FSHR (asparagine-173; numbering includes leader sequence) to glutamine resulted in a complete loss of cell-surface rat LH receptor;, detergent extraction of cells did not reveal a trapped form of the receptor [55]. Mutation of Asn-291, the other conserved glycos3'lation site, had no effect. Mutation of asparagine-77 and 152 ~esnlted in a reduction of the nambcr of binding sites with no change in affinity [55]. These ~ o important observations underscore the concept that these receptors may have quite different structure-function attributes despite similar sequences. It remains to be determined whether ali sLx potential sites on these receptors are giycosylated. Similar work with follitropin remains to be done, and a comparison between species seems warranted. IV-C. Transmembrane and cytoplasmic donmins and signal transduction TSH, L H and FSH receptors The large extracellular domain of the glycoprotein hormone re--'eptor~ is dhTerent frot~ the short extracellular domain of other G-protein coupled receptors. Analogy with the G-protein coupled receptors with short extracellular domains suggests that part of the effector site resides in the extracellular portions of the transmembrane regions. It was of interest that antipeptide antibodies against the first, second and third loops of the putative transmembrane domain did not block TSH binding to receptor, but antibodies against the second and third loops stimulated cAMP production [56]. In addition, hCG bound with low affinity to truncated rat LH receptors, containing only 10 amino acids of the extracellular domain, and stimulated cAMP production [57]. Such evidence suggests tha~. consideration must be given to a model in which the glycoprotein hormone interacts directly with the extracellular loops of the transmembrane region in addition to a high-affinity interaction with *,he large extracellular domain. The charge of the amino acids within each putative transmembrane segment may also be important for signal transduction since mutation of aspartic acid-383 of the LH receptor to a glutamine resulted in a loss of high affinity binding [58]. The phenomenon of desensitization may involve the cytoplasmic tail of the receptor. Treatment of Leydig tumor cells with antisense oligonucleotides that code for the third extracellular loop of the rat LH receptor produced truncated receptor and prevented desensitization induced by LH, PMA or BtecAMP [22]. The'=e
results suggest that the intracellular cytoplasmic tail of the LH receptor is required for the desensitization process to occur, possibly through phosphorylation of sites on the C-terminal cytoplasmic end of the receptor. The three cytoplasmic loops and the C-terminal tail of the receptors are potential sites of interactions with the G-psoteins. Mutations in the first cytoplasmic loop and the carboxyl ends of the second and third cytoplasmic loops of TSH receptor cause a loss of signal transduction [59]. The Coterminal region 709-764 is not needed for "ISH action. Mutations in the amino terminus of the second loop, and in the C-terminal end of the third loop, resulted in a decrease in affinity of ligand binding which made it difficult to interpret a decrease in signal transduction. Attempts to further define specific amino acids in the C-terminal end of the third cytoplasmic loop revealed that no single residue was important for signal transductlon [60]. These data support the concept that the cytoplasmic domains of the receptor are involved in signal transduction. V. Futare d i r ~ The cloning of the cDNAs for the glycoprotein receptors has allowed deduction of protein sequence information from which reasonable hypotheses about siru~i,~re and function could be formulated. Elucidation of the primary structures of the glycoprotein hormones can be used as an example of how such information led to important structure and function correlates. However, in the case of the hormones, as will be the case with the receptors, the information gained by such studies can only pinpoint the sequences, and specific amino acids, involved in functional attributes. It should be clear from the foregoing discussion that hormone binding by the receptor involves discontinuous sequences. Whether these sequences are assembled, to form a discontinuous epitope will require ~ r t b e r tertiary analysis. One approach would use monoclonal antibodies prepared by immunizing with the receptors. These antibodies can be epitope mapped with synthetic or recomb',ant receptor peptides in order to determine the dis~ontinuous sequences which form regional su~aces on the receptors. It may be that the binding determinants of both the giycoprotein hormones and their receptors which c6nfor 'specificity', depend on conformatioaal diversity (in ~ e face of sequence conservation). If so, the three-dimensional structures of each hormone and receptor will be required to develop a better understanding of this interaction. Of course, since it is quite possible that c~nformatiou~l changes may occur as a result of the binding interaction, the three dimensional structure of the complex as well as the individual proteins will have to be determined. It would be desirable to
293 formulate h y p o t h e s e s b a s e d o n a t h r e e dimensional structure r a t h e r t h a n trying to a p p r o x i m a t e c o n f o r m a tional attributes. T h e t h r e e d i m e n s i o n a l s t r u c t u r e o f the g r o w t h h o r m o n e - r e c e p t o r complex [61] h a s b e e n d e t e r m i n e d . Suitable high level expression o f insulin r e c e p t o r [62], L H r e c e p t o r [15] a n d o t h e r G - p r o t e i n c o u p l e d r e c e p t o r s [63] in insect cells h a s b e e n achieved. T h u s , t h e p r o d u c t i o n o f sufficient p r o t e i n to p e r f o r m crystal!ixation trials a n d ultimately to allow crystallization o f t h e reclzptors s h o u l d b e a n a r e a o f high priority for investigators o f t h e glycoprotein h o r m o n e r e c e p t o r s a n d t h e i r ligands. T h e level o f r e c e p t o r p r o t e i n o n the cell s u r f a c e a p p e a r s to be subject t o r e g u l a t i o n a t several levels, including g e n ¢ expression, a n d possibly proteolysis a n d p h o s p h o w l a t i o n . F u t u r e investigations will b e challenging since e a c h o f these p a r t i c u l a r m e c h a n i s m s for regulatinu im, olves incompletely u n d e r s t o o d hierarchical complex r e g u l a t o r y c o m p o n e n t s . Yet n e w a n d i m p o r t a n t i n f o r m a t i o n h a s a l r e a d y b e e n g a i n e d f r o m early w o r k in this a r e a . Analysis o f p r o m o t e r regions u p s t r e a m o f t r a n s c r i p tional s t a r t sites suggests t h a t up- a n d d o w n - r e g u l a t i o n o f t h e glycuprotein h o r m o n e r e c e p t o r g e n e s m a y involve b o t h c A M P - d e p e n d e n t a n d - i n d e p e n d e n t m e c h a nisms. Certainly, t h e c A M P down*regulation o f r e c e p t o r g e n e expression is a n interesting a r e a o f study. M u c h p r o t e i n chemistry will n e e d t o b e d o n e to identify w h e t h e r p h u s p h o r y l a t i o n o f e a c h o f t h e t h r e e glycoprotein h o r m o n e r e c e p t o r s o c c u r s a n d w h e t h e r desensitization is a c o m m o n p a t h w a y t o a t t e n u a t i o n o f cellular response. W h e t h e r o r n o t all potential glycosylation sites h a v e c a r b o h y d r a t e , a n d t h e c a r b o h y d r a t e s t r u c t u r e s p r e s e n t , a s well a s the diversity o f glycosylation a n d complexity b e t w e e n t h e receptors, as well as b e t w e e n species, will also n e e d t o b e d e t e r m i n e d . T h e clinical a n d p u b l i c h e a l t h i m p a c t o f w o r k to d a t e a n d f u t u r e w o r k is likely to b e great. A n obvious e x a m p l e is t h e identification o f the d e t e r m i n a n t s o f a u t o i m m u n i t y in Graves" disease. It c a n b e e x p e c t e d t h a t n e w u n d e r s t a n d i n g s o f diseases o f thyroid function a n d o f infertility will b e f o r t h c o m i n g . In addition, n e w a p p r o a c h e s to fertility c o n t r o l m a y also result. Acknowledgement T h i s study w a s s u p p o r t e d b y N I H H D 1 8 4 0 7 . References I pierce, J. and Parsons, T.F. (1981) Annu. Rev. Biochem. 50. 465-495. 2 Baenziger, J.U. and Green, E.D. (1988) Biochim. Biophys. Acta 947, 287-306. 3 Green, E.D. and Baenziger, J.U. (19881 J. Biol. Chem. 263. 25-35. 4 Dias, J.A. (1992.)Trends EndocrinoL Metabo 3, 24-29.
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