TiBS 17 - DECEMBER 1992
THE ROI.F. OF INOSITOL PHOSPHATES in hormone-dependent increases in intracellular free calcium (Ca2÷) set the stage for identifying the pivotal role of phospholipids and phospholipases as sources and regulators, respectively, of second messenger molecules. The bestcharacterized system remains the regulated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], The production of two second messengers from this molecule by the action of specific phospholipase C (PLC) enzymes can be stimulated by growth factors whose receptors are linked to tyrosine kinase activity, hormones which act through G protein-linked receptors, the concentration of intracellular Ca2÷ and other agents of undefined mechanism. The participation of G proteins in the regulation of this activity was initially suggested by specific requirements for guanine nucleotides for stimulation by hormones and the more direct effects of non-hydrolysable guanine nucleotides and aluminium fluoride (AI~, a universal activator of the heterotrimeric G proteins), An additional reagent that implicated a role for G proteins was the toxin from Bordatella pertussis, which ADP-ribosylates the cz-subunits of G~ and Go proteins and attenuates their function. The action of several hormones was abolished by this toxin, but in most cases, the toxin had little or only partial effect, This indicated that a unique G protein (frequently referred to as Gp) was involved in this latter stimulation (for reviews see Refs 1, 2). This review focuses on more recent experiments and their impact on our understanding of G protein-linked regulation of phosphatidylinositol-specific phospholipase C (Ptdlns-PLC).
Regulation of phospholipase C by Gproteins Specific phospholipase C enzymes can hydroiyse phosphatidylinositol 4,5bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the Gq subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the Gq subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.
mary activity of the G protein, Thus, favored, Stimulation of the G protein Gs~ stimulates adenylate cyclase and results when it binds GTP rather than transducin ~ (Gt~¢) activates a cyclic GDP. Receptors interact most efficiently GMP-dependent phosphodiesterase in with the heterotrimeric form of the G retina, protein and accelerate activation by The G1 proteins were identified as increasing the rate of dissociation of inhibitory regulators of adenylate cyc- GDP and potentially enhancing associlase. More recently, the Gi and Go pro- ation of GTP. When activated, the affinity teins have been implicated in the regu- between the ~- and ~7-subunits of the G lation of several ion channels, The Gt, G1 protein is decreased, This increases the and Go proteins are substrates for per- likelihood of dissociation of subunits and tussis toxin (PTX); attenuation of hor- the generation of two potential pathmone action by this toxin implicates the participation of one or more of these proteins. Several new ~-subunits that are not substrates for PTX have been recently identified by either purification of proteins or isolation of homologous cDNAs (¢Zq, GDP "~_, " czn_16), Several of these are discussed below. The G proteins and activation G proteins and their funcThe G proteins that mediate regu- tions have been reviewed lation of several effector molecules by extensively (see Refs 3-8 hormones constitute a large family of for examples). highly homologous proteins. The proA short discussion of teins are heterotrimers consisting of cz-, the G protein cycle of acti~- and ~subunits. The ~-subunits ap- vation is presented here pear to be most diverse and have been to facilitate subsequent distraditionally used to define the purified cussion. Numerous experheterotrimeric proteins, Frequently, the iments support a simple Figure 1 Model for activation of G proteins. The functional state cz-subunits can also account for the pri- model for the activation of a G protein is determined by its bound nucleotide. of G proteins (Fig. 1). In With GDP, the G prOtein is inactive and subunit asthe basal state, the cz-subP. C. Sternweis and A. V. Smrcka are at the . . . . . . . . . . is favored. With bound GTP,the G protein is unit contains bound GDP, Department of Pharmacology, K5.100, activated and the affinity between its o~- and 13~ and association of ~-and Universityof TexasSouthwestern Medical subunits is markedly reduced. Receptors stimulate G Center, Dallas, TX 75235, USA. 137-subunlts is highly pr(~teins by catalysing exchangeof GTPfor GDP. © 1992,ElsevierSciencePublishers,(UK) 502
TIBS 3_7 - DECEMBER1992
those deduced from two highly homologous cDNAs, G~family eCq and c£i (Ref. 10). The ~z preparation was subseGo family quently shown to stimu(X,q + 48 late PLC from brain u. At C(,11 4the same time, a G pro57 0('14 4" tein immunologically re0(15 ? lated to cz~ was isolated C~16 + from bovine liver based on its ability to activate a G~2family PLC activity12. This prep(~12 o 65 aration, which also con(~13 ') tains CZqand uu, was used to demonstrate G protein Figure 2 regulation of a ]3, but not Relationship among known PTX-insensitive G orotein a y or 5 isotype of PtdIns(z-subunits. The names and members of the subfamilies PLC13.A G protein isolated follow the assignment suggested by Simon and colfrom turkey erythrocytes leagues5. Numoers indicate the percentage amino acid identity between subunits or the average identity has also been shown to among groups. A plus sign indicates that the (z-substimulate PLC from erythunit has been shown to activate Ptdlns-PLC (Refs in rocytes TM. The absolute text; A. G. Gilman, P. Sternweis and colleagues, unidentity of the avian G propublished). tein remains to be determined, but it is immunologically related to ~qm. ways [¢z(GTP) and free [~y-subunitsl for Further experiments have demondownstream regulation. Finally, the G strated that members of the Gq family protein ~-subunit has an intrinsic hy- can interact with appropriate receptors drolytic activity that slow137 (rates
THE ROI.F. OF INOSITOL PHOSPHATES in hormone-dependent increases in intracellular free calcium (Ca2÷) set the stage for identifying the pivotal role of phospholipids and phospholipases as sources and regulators, respectively, of second messenger molecules. The bestcharacterized system remains the regulated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], The production of two second messengers from this molecule by the action of specific phospholipase C (PLC) enzymes can be stimulated by growth factors whose receptors are linked to tyrosine kinase activity, hormones which act through G protein-linked receptors, the concentration of intracellular Ca2÷ and other agents of undefined mechanism. The participation of G proteins in the regulation of this activity was initially suggested by specific requirements for guanine nucleotides for stimulation by hormones and the more direct effects of non-hydrolysable guanine nucleotides and aluminium fluoride (AI~, a universal activator of the heterotrimeric G proteins), An additional reagent that implicated a role for G proteins was the toxin from Bordatella pertussis, which ADP-ribosylates the cz-subunits of G~ and Go proteins and attenuates their function. The action of several hormones was abolished by this toxin, but in most cases, the toxin had little or only partial effect, This indicated that a unique G protein (frequently referred to as Gp) was involved in this latter stimulation (for reviews see Refs 1, 2). This review focuses on more recent experiments and their impact on our understanding of G protein-linked regulation of phosphatidylinositol-specific phospholipase C (Ptdlns-PLC).
Regulation of phospholipase C by Gproteins Specific phospholipase C enzymes can hydroiyse phosphatidylinositol 4,5bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the Gq subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the Gq subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.
mary activity of the G protein, Thus, favored, Stimulation of the G protein Gs~ stimulates adenylate cyclase and results when it binds GTP rather than transducin ~ (Gt~¢) activates a cyclic GDP. Receptors interact most efficiently GMP-dependent phosphodiesterase in with the heterotrimeric form of the G retina, protein and accelerate activation by The G1 proteins were identified as increasing the rate of dissociation of inhibitory regulators of adenylate cyc- GDP and potentially enhancing associlase. More recently, the Gi and Go pro- ation of GTP. When activated, the affinity teins have been implicated in the regu- between the ~- and ~7-subunits of the G lation of several ion channels, The Gt, G1 protein is decreased, This increases the and Go proteins are substrates for per- likelihood of dissociation of subunits and tussis toxin (PTX); attenuation of hor- the generation of two potential pathmone action by this toxin implicates the participation of one or more of these proteins. Several new ~-subunits that are not substrates for PTX have been recently identified by either purification of proteins or isolation of homologous cDNAs (¢Zq, GDP "~_, " czn_16), Several of these are discussed below. The G proteins and activation G proteins and their funcThe G proteins that mediate regu- tions have been reviewed lation of several effector molecules by extensively (see Refs 3-8 hormones constitute a large family of for examples). highly homologous proteins. The proA short discussion of teins are heterotrimers consisting of cz-, the G protein cycle of acti~- and ~subunits. The ~-subunits ap- vation is presented here pear to be most diverse and have been to facilitate subsequent distraditionally used to define the purified cussion. Numerous experheterotrimeric proteins, Frequently, the iments support a simple Figure 1 Model for activation of G proteins. The functional state cz-subunits can also account for the pri- model for the activation of a G protein is determined by its bound nucleotide. of G proteins (Fig. 1). In With GDP, the G prOtein is inactive and subunit asthe basal state, the cz-subP. C. Sternweis and A. V. Smrcka are at the . . . . . . . . . . is favored. With bound GTP,the G protein is unit contains bound GDP, Department of Pharmacology, K5.100, activated and the affinity between its o~- and 13~ and association of ~-and Universityof TexasSouthwestern Medical subunits is markedly reduced. Receptors stimulate G Center, Dallas, TX 75235, USA. 137-subunlts is highly pr(~teins by catalysing exchangeof GTPfor GDP. © 1992,ElsevierSciencePublishers,(UK) 502
TIBS 3_7 - DECEMBER1992
those deduced from two highly homologous cDNAs, G~family eCq and c£i (Ref. 10). The ~z preparation was subseGo family quently shown to stimu(X,q + 48 late PLC from brain u. At C(,11 4the same time, a G pro57 0('14 4" tein immunologically re0(15 ? lated to cz~ was isolated C~16 + from bovine liver based on its ability to activate a G~2family PLC activity12. This prep(~12 o 65 aration, which also con(~13 ') tains CZqand uu, was used to demonstrate G protein Figure 2 regulation of a ]3, but not Relationship among known PTX-insensitive G orotein a y or 5 isotype of PtdIns(z-subunits. The names and members of the subfamilies PLC13.A G protein isolated follow the assignment suggested by Simon and colfrom turkey erythrocytes leagues5. Numoers indicate the percentage amino acid identity between subunits or the average identity has also been shown to among groups. A plus sign indicates that the (z-substimulate PLC from erythunit has been shown to activate Ptdlns-PLC (Refs in rocytes TM. The absolute text; A. G. Gilman, P. Sternweis and colleagues, unidentity of the avian G propublished). tein remains to be determined, but it is immunologically related to ~qm. ways [¢z(GTP) and free [~y-subunitsl for Further experiments have demondownstream regulation. Finally, the G strated that members of the Gq family protein ~-subunit has an intrinsic hy- can interact with appropriate receptors drolytic activity that slow137 (rates
