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The role of prenylation in G-protein assembly and function
Cell. Signal. Vol. 8, No. 6, pp. 433-437, 1996 Copyright © 1996 Elsevier Science Inc.
ISSN 0898-6568/96 $15.00 PII S0898-6568(96)00071-X ELSEVIER
The Role of Prenylation in G-Protein Assembly and Function Joyce B. Higgins* and Patrick J. Casey DEPARTMENTS OF MOLECULAR CANCER BIOLOGY AND BIOCHEMISTRY,
DUXE UNIVERSITYMEDICALCENTER,DURHAMNC 27710-3686 USA
ABSTRACT. Heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are vital components of numerous signal transduction pathways, including sensory and hormonal response systems. G-proteins transduce signals 8om heptahelical transmembrane receptors to downstream effectors.The localizationof a G-protein to the plasma membrane, as well as its interaction with the appropriate receptor and effector, are essential for its function. In addition, the association of a G-protein's subunits to form its trimer is required for interaction with its receptor. The G-protein y subunits (G~) are subject to a set of carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and methylation. Recent advances which elucidate the contributions that the post-translational modifications of the Gv subunit have on the assembly, membrane association, and function of the G-protein trimer reveal that these modifications are required for important protein-protein, in addition to membrane-protein, interactions. Copyright ©/996 Elsevier Science Inc. CELLSmNAL8;6:433-437, 1996. KEY WORDS. G-protein, Prenylation, Isoprenylation, Subunit interaction, Processing, Famesyl, Geranylgeranyl, Methylation
INTRODUCTION Guanine nucleotide-binding regulatory proteins (G-proteins) are signal mediators in a number of pathways that involve the heptahelical family of transmembrane receptors and a variety of downstream effectors [1, 2]. G-proteins comprise three subunits, denoted G~, G~, and G~, which in the inactive state form a heterotrimer, designated G ~ . G-proteins transduce extracellular stimuli into cellular changes through a common mechanism. Association of the heterotrimer with a ligand-activated heptahelical receptor stimulates a conformational change in G~, allowing the release of guanosine diphosphate (GDP) and subsequently the binding of guanosine triphosphate (GTP). The G~ and G~ subunits dissociate from the GTP-bound G~ as a tight complex (G~). Both the free G ~ complex and the activated G~-GTP are able to act independently, synergistically, or antagonistically on specific effector molecules to alter second-messenger concentrations and thus alter specific cellular processes. The hydrolysis of the bound GTP to GDP and the release of P~ leads to inactivation of G~, re-association of the G~ with G~, and finally re-coupling of the heterotrimer to the receptor if the agonist is still present. Nearly all the players in these signalling processes are localized to the plasma membrane, either through peripheral or integral membrane association [14]. * Address all correspondence to Joyce B. Higgins, Department of Chemistry, Eastern Illinois University, Charleston, IL 61920. Received 6 January; and accepted 28 February 1996.
Although membrane association of G-proteins is essential for their biological activity, none of the three subunits contains a transmembrane-spanning domain to account for the membrane interactions. Recent discoveries have revealed that essentially all of the G proteins are instead modified by covalently attached lipid moieties [4-6]. Three types of lipids have been found covalently linked to G-protein subunits (Fig. 1). The G~ subunits are modified by myristate, a 14-carbon saturated fatty-acyl group; palmitate, a 16-carbon fatty-acyl chain; or by both groups. The myristate is linked to G~ through an amide bond to the glycine residue at the amino terminus [7]. This modification occurs co-translationally and is chemically stable to all but strong acid. Palmitate, on the other hand, is post-translationally attached to G~ through a base-labile thioester bond to the cysteine residue(s) near the amino terminus [8, 9]. Palmitoylation is a reversible modification whose lability may play a dynamic role in signal-transduction regulation [5, 10]. The G~ subunits of G-proteins contain the so-called -CaaX motif at their carboxyl terminus, and are modified by a process termed prenylation. This post-translational modification includes transfer of an isoprenoid lipid from a prenyldiphosphate substrate to the invariant cysteine of the -CaaX motif by specific protein prenyltransferases [11]. Proteins can be modified by one of two distinct isoprenoids, the 15-carbon famesyl or the 20-carbon geranylgeranyl. The G~I subunit associated with the retinal G-protein is famesylated [12, 13], as is an additional, recently discovered Gy
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well as a prenyl moiety, it has been proposed that the molecule may be involved in the trafficking of prenylated proteins to the intracellular membrane, where the proteolysis and methylation occur. The lipid modifications of the G-protein subunits not only contribute to membrane interaction of the proteins, but apparently also contribute to critical protein-protein interactions in the signalling pathways they control. This review focuses on the contributions of the G~ modifications to the assembly, membrane association, and function of trimeric G-proteins.
HETEROTRIMER FORMATION
FIGURE 1. Lipid modifications of G-proteins. The three types of lipid modifications found on G-proteins are depicted here on the basal state of the heterotrimer associated with the plasma membrane. The G~ subunit depicted contains both a thioesterlinked palmitoyl group and a myristoyl group attached via an amide bond to the N-terminal glycine. The G~ subunit contains a geranylgeranyl isoprenoid linked through a thioether bond to a cysteine residue that also contains a methylester. While all the lipids are shown inserted into the membrane bilayer, the existence of additional membrane proteins involved in the membrane association has not been ruled out.
[14], whereas the remainder of known G~ subunits are modified by the geranylgeranyl moiety [14-16]. As with essentially all -CaaX type proteins, prenylation is followed by cleavage of the last three residues of Gy by an endoprotease, and finally the carboxyl terminal prenylcysteine is methylated by a methyltransferase [11, 17]. While the first two processing events (i.e., prenylation and proteolysis) are stable modifications, the methylester bond is somewhat labile. Thus, the methylation state of prenylated proteins may play a dynamic role in the regulation of signaltransduction pathways [18, 19]. Enzymes capable of both the methylation and demethylation of prenylated proteins have been identified in retinal membranes [20]. Another potential player in G~ processing is a binding protein that has been identified in the microsomal membrane fraction, and which recognizes both farnesylated and geranylgeranylated proteins [21]. Since high-affinity binding to this site requires the last three amino acids of the -CaaX motif as
As noted earlier, G-proteins exist as trimers in the basal state, and G~ dissociates from the GTP-bound G~ subunit upon activation. The extremely stable G~ dimer is also resistant to proteolysis [22]; this property has been exploited in studies assessing G~ dimer assembly. Even after cleavage at this single site, the G~ and Oysubunits remain tightly associated without any disulfide bonds; surprisingly, the cleaved complex can still form a functional trimer with G~ [23]. The first successful reconstitution of G~y dimers was accomplished by assembling G~ and G~ proteins translated in rabbit reticulocyte lysates [24]. Further studies demonstrated that reconstitution was dependent on the system utilized for the expression of G~, but not that for G~ [24, 25]. Gy expressed and purified from systems as evolutionarily divergent as bacteria, insects, and animals are all competent for in vitro assembly [24-26]. However, while G B subunits expressed in both insect cells and rabbit reticulocyte lysates are competent for in vitro assembly [24, 25], those expressed in wheat-germ extracts or Escherichia coli are not [25, 27]. Formation of a stable dimer by co-expression of G~ and G~ in vivo also depends on the cell type; co-expression in COS cells and insect cells produces stable dimers [24, 28-31], whereas co-expression in bacteria is not suitable for G~ dimer formation [25]. The expression conditions required to produce or reconstitute stable G~y dimers indicate that an accessory protein or proteins may facilitate the assembly reaction or prevent G~ aggregation prior to assembly. The assembly of G~y dimers is most probably a cytosolic event that occurs prior to the processing of the G~ subunit [25, 26]. Substitution of Cys6s in G~2 (the site of isoprenoid addition) with Ser prevents the prenylation of his polypeptide, and its co-expression with G~ results in production of a cytosolic G~ complex [29, 32]. These studies demonstrated that the assembly of G~ dimers is not dependent on prenylation of G~. Reconstitution of G~ dimers utilizing G~ expressed in Sf9 cells and G~ purified from E. coli has provided additional information in this regard. Unmodified and prenylated G~ subunits can assemble with G~, but the unmodified form is more efficient [26]. These data confirm that prenylation is not required for G~ assembly. On the other hand, the truncated-prenylated G~ protein (i.e., G~ that is missing the "-aaX" residues of the -CaaX motif) does not assemble with G~, suggesting that assembly must occur before proteolysis [25]. The fully processed G~ subunit is
Prenylation in G-protein Assembly and Function
435
LaaLi FIGURE 2. Model for assembly and processing of Gpv. In this model, the G~ and Gv proteins synthesized in the cytosol associateto form stable G~v dimers. The soluble Gp~ complex thus formed serves as a substrate for enzymatic prenylation of the Gv polypeptide. The prenylated dimer then translocates to the microsomal membrane through associating with a specific binding protein, whereupon the processing is completed by actions of an endoprotease that cleaves the three C-terminal residues, followed by methylation of the exposed prenylcysteine. The mature protein is then targeted to the plasma membrane by mechanisms even less clear.
CaaX
I
even less efficient in assembly with G~ than the Gv subunit that is only prenylated, and G~ complexes containing an unmodified G~ serve as excellent substrates for the protein prenyltransferase, demonstrating that prenylation can occur after complex assembly [26]. Overexpression of Gy subunits in COS cells results in an accumulation of Gv-proteins on an intracellular membrane, perhaps because the proteolysis or methylation of the prenylated proteins, which occurs on intracellular membranes, becomes rate-limiting, and overexpression of G~ and Oysubunits in the same cell shows accumulation of both subunits at the intracellular membrane [29]. Taken together, these results indicate that processing of Gy most probably occurs after assembly of the G~s complex. The association of G~ and G~ into the trimeric G ~ is necessary not only to attenuate signalling of both the activated G~ and the free G~v complex, but also for recognition of the G~ subunit by the receptor and for enhanced membrane association (see below). Myristoylation of G~ subunits enhances their interaction with G~ [33]. Although prenylation of G~ is not required for the assembly of G~ complexes, the G~ complex does require a prenylated Gv for high-affinity interaction with G~ [25, 34]. Whether the requirements for lipid modifications of both G~ and G, for
Protein Prenyltransferasa •.W.,J.~P ~ ~
NH3+
high-affinity trimer formation reflect an interaction of these lipids in the assembled species is not yet clear. Methylation of the carboxyl-terminal prenylcysteine in Gyl increases the interaction of G,~ with O131y] by 2-fold over the unmethylated G~v isoform [19, 35]. In addition, the Gt~G~ interaction can be inhibited by peptides corresponding to the carboxyl terminus of Gvl, but only if the peptide is farnesylated. Substituting geranylgeranyl for the famesyl group on the peptide increases the inhibition by 2-fold, whereas methylation of the farnesylated peptide increases the inhibition by 3-fold [35]. These data suggest that the degree of hydrophobicity of the G~I subunit plays a significant role in these subunit interactions. Thus, results with a wide variety of experimental approaches indicate that prenylation and subsequent processing of G~, in addition to the acyl modifications of G~, contribute to the stability of the G~O~y interaction. MEMBRANE ASSOCIATION The localization of the G-proteins to the intracellular surface of the plasma membrane increases their effective concentrations and thus facilitates their interactions with both
436 receptors and effectors. Palmitoylation, myristoylation, and Glay interaction all contribute to the membrane association of G~ [4-6]. The contribution of G~ was first demonstrated through the use of reconstitution studies. Efficient association of bovine brain G,~ with phospholipid vesicles was found to require the presence of G~, on the vesicles [36]. This is not an all-or-none event, however, since G~ subunits over-expressed in COS cells are almost exclusively found in the membrane fraction even when the total amount of G~ is in excess of the G~y concentration [37]. It seems likely that the presence of membrane-associated G~ in the cells is necessary to target O~ to the membrane, but is not essential to keep the G~ associated with the plasma membrane once it has encountered the lipid bilayer. A variety of approaches have been used to demonstrate that the membrane association of the G~s complex is predominantly the consequence of the post-translational modification of the Gy subunit. Prenylation of the G~ protein promotes the membrane association of both G~ monomers and G~ dimers [28, 29]. Co-expression of G~ and Gs increases the fraction of Gy that is prenylated and subsequently membrane-associated [30, 38], presumably through an increased stability of G~ when it has assembled with G~. The localization of the G~ complex in cells can be altered by treating the cells with the drug compactin. Compactin is an inhibitor of hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway, and treatment of cells with the drug is a technique commonly used to prevent isoprenoid synthesis and thus protein prenylation. Following treatment of cells with compactin, a fraction of the G~ complexes became cytoplasmic, while the bulk of the G~ remained membrane-associated. Membrane association of G~ could be restored by supplementing compactin-treated cells with exogenous mevalonate, the product of HMG-CoA reductase [29]. Additionally, as noted above, the C68S G~ mutant has a cytoplasmic distribution in Sf9 cells, and its co-expression with G~ results in production of a cytosolic G~ complex [29, 32]. These results demonstrate that prenylation of Gy is essential for membrane association of the G~ complex. The consequences of the proteolysis and methylation steps in G~ processing with regard to membrane association of the G~ complex have been more difficult to address, since these enzymatic steps occur quite rapidly after prenylation. While G~ complexes isolated from a number of different tissue sources apparently contain only methylated G~ [39], retinal G~ complexes are found as a mixture of complexes containing both unmethylated and methylated G~ [40, 41]. Separation of these two forms revealed that the rnethylated form has a higher affinity for the retinal membranes than the unmethylated form. Moreover, the presence of G~ significantly enhanced the association of the methylated G~ to the membrane, but only slightly increased the membrane association of the unmethylated form [40]. Similar results have been obtained using G~s complexes that were demethylated by esterase treatment after purification [42]. These data indicate that the methyl-
J. B, Higgins and P. J. Casey ation state not only increases the affinity of G~ for membranes but also its affinity for G~, and also that membrane binding of G~ is influenced by G~. Additional information on interaction of the G-protein subunits with the lipid bilayer has come from reconstitution studies using phospholipid vesicles of defined composition, which have provided evidence that, in addition to the lipid modifications, charged amino acid domains also contribute to the membrane association of both the G~ complex and G~ subunit [35]. These data have also been interpreted as suggesting that the domains responsible for the membrane attachment of the individual subunit complexes (i.e., G~, G~-GTP) are different from that of the heterotrimer.
G PROTEIN FUNCTION After years of languishing in obscurity, the G~ complex has finally received its rightful share of attention for active roles in G-protein function. Since 1987, numerous protein targets that are regulated by G~ have been identified [43, 44]. The elucidation of multiple activities for the G~ complex has transformed the classical view of G-protein signalling and has emphasized the importance of understanding the mechanisms of G~ interactions. The recognition of the heterotrimer by the receptor is one of the first steps in the signalling pathway. G-proteincoupled receptors appear to be more discriminating than G~ subunits with regard to their interaction with specific G~ dimers. While the G,~ subunit will bind G~ complexes composed of G~I and a number of G~ isotypes, coupling of the transducin heterotrimer to its receptor, rhodopsin, is more efficient with the trimer containing the Gs~ isotype [45]. Metarhodopsin II, the receptor conformation that is stabilized by the binding of transducin, requires prenylated G~, and preferentially binds transducin containing the methylated Gyl subunit [19, 40]. In fact, a farnesylated peptide corresponding to the C-terminus Gyl can uncouple the transducin trimer from the receptor and stabilize metarhodopsin II [46], and the farnesyl group itself appears to be a critical determinant in this interaction [47]. The preference of both the receptors and the G~ subunits for the processed cysteine on G~s indicates that the modifications of the Gs subunit contribute to protein-protein interactions essential to G-protein function. As noted above, the list of effector molecules that are regulated by G~ is growing, and many of these effectors can be regulated not only by G~ but by Go as well. A particularly well-studied example is the regulation of adenylyl cyclase. Some isotypes of this enzyme are inhibited by the G~ complex, while others are synergistically stimulated by G~ in conjunction with activation by G,~ [48]. The effects of the G~ complex on both classes of adenylyl cyclase require processed G~ [34]. Another effector that has been extensively studied is the 132 isotype of phospholipase C (PLCI3/); this protein is independently activated by both the G~ and G~ subunits [34, 49]. Prenylation of the G~ subunit is necessary for the activation of PLC[~2 by G~ in intact cells and in re-
Prenylation in G-protein Assembly and Function constituted phospholipid vesicle systems [50], and the effect is enhanced by methylation of the prenylcysteine [18]. Although the precise contributions of G~ processing to effectorG~v interactions have not been fully analyzed, particularly the respective roles of enhanced membrane association versus a direct influence on protein-protein interactions, current observations indicate that the processing of the G~ subunit in the O~y complex is essential for G ~ regulation of most effector molecules. CONCLUSIONS Signal-transduction pathways mediated by trimeric G-proteins are numerous, and the interactions of the G-protein subunits with the components of these systems are critical determinants of their activity and specificity. The mechanism by which the G-protein subunits interact with each other, with other signalling molecules, and with the membrane surface are of definite import. Although the contributions of the covalent modifications of the G~ and G~ subunits are significant, they are certainly not the sole determinants of these subunits' effects. Nonetheless, an increased awareness of the importance of lipid modifications in these processes should lead to a more detailed description of the molecular events involved in these signalling processes. Dr. Casey is an establishedinvestigatorof the American Heart Association.
References 1. Neer E. J. (1995) Cell 80, 249-257. 2. Gilman A. G. (1995) Angew. Chem. Int. Ed. Engl. 34, 14061419. 3. Dolman H. G., Thomer J., Caron M. G. and Lefkowitz R. J. (1991) Annu. Rev. Biochem. 60, 653-688. 4. Casey P. J. (1994) Curr. Opin. Cell. Biol. 6, 219-225. 5. Wedegaertner P. B., Wilson P. B. and Bourne H. R. (1995)J. Biol. Chem. 270, 503-506. 6. Milligan G., Parenti M. and Magee A. I. (1995) Trends Biochem. Sci. 20, 181-187. 7. BussJ. E., Mumby S. M., Casey P. J., Gilman A. G. and Sefton B. M. (1987) Proc. Natl. Acad. Sci. USA 84, 7493-7497. 8. Parenti M., Vigano M. A., Newman C. M. H., Milligan G. and Magee A. I. (1993) Biochem. J. 291,349-353. 9. Linder M. E., Middleton P., Hepler J. R., Taussig R., Gilman A. G. and Mumby S. M. (1993) Proc. Natl. Acad. Sci. USA 90, 3675-3679. 10. Degtyarev M. Y., Speigel A. M. and Jones T. L. Z. (1993) J. Biol. Chem. 268, 23769-23772. 11. Zhang F. L. and Casey P. J. (1996) Annu. Rev. Biochem. 65, 241-269. 12. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T. and Shimonishi Y. (1990) Nature 346, 658-660. 13. Lai R. K., Perez-Sala D., Canada F. J. and Rando R. R. (1990) Proc. Natl. Acad. Sci. USA 87, 7673-7677. 14. Ray K., Kunsch C., Bonner L. M. and Robishaw J. D. (1995) J. Biol. Chem. 270, 21765-21771. 15. Mumby S. M., Casey P. J., Gilman A. G., Gutowski S. and Stemweis P. C. (1990) Proc. Natl. Acad. Sci. USA 87, 58735877. 16. Yamane H. K., Famsworth C. C., Xie H., Howald W., Fung B. K.-K., Clarke S., Gelb M. H. and Glomset J. A. (1990) Proc. Natl. Acad. Sci. USA 87, 5868-5872.
437 17. Clarke S. (1992) Annu. Rev. Biochem. 61,355-386. 18. Parish C. A., Smrcka A. V. and Rando R. R. (1995) Biochemistry 34, 7722-7727. 19. Fukada Y., Matsuda T., Kokame K., Takao T., Shimonishi Y., Akino T. and Yoshizawa T. (1994) J. Biol. Chem. 269, 51635170. 20. Perez-Sala D., Tan E. W., Canada F. J. and Rando R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 3043-3046. 21. Thissen J. A. and Casey P. J. (1993) J. Biol. Chem. 268, 13780-13783. 22. Winslow J. W., Van Amsterdam J. R. and Neer E. J. (1986) J. Biol. Chem. 261, 7571-7579. 23. Thomas T. C., Sladek T., Yi F., Smith T. and Neer E. J. (1993) Biochemistry 32, 8628-8635. 24. Schmidt C. J. and Neer E. J. (1991) J. Biol. Chem. 266, 45384544. 25. Higgins J. B. and Casey P. J. (1994)J. Biol. Chem. 269, 90679073. 26. Higgins J. B. Ph.D. Thesis. Duke University, 1995. 27. Mende U., Schmidt C. J., Yi F., Spring D. J. and Neer E. J. (1995) J. Biol. Chem. 270, 15892-15898. 28. Simonds W. F., Butrynski J. E., Gautam N., Unson C. G. and Spiegel A. M. (1991) J. Biol. Chem. 266, 5363-5366. 29. Muntz K. H., Sternweis P. C., Gilman A. G. and Mumby S. M. (1992) Mol. Biol. Cell 3, 49-6l. 30. Robishaw J. D., Kalman V. K. and Proulx K. L. (1992) Biochem J. 286, 677-680. 31. Graber S. G., Figler R. A., Kalman-Maltese V. K., Robishaw J. D. and Garrison J. C. (1992) J. Biol. Chem. 267, 1312313126. 32. Dietrich A., Meister M., Spicher K., Schultz G., Camps M. and Gierschik P. (1992) FEBS Lett. 313, 220-224. 33. Linder M. E., Pang I-H., Duronio R. J., Gordon J. I., Stemweis P. C. and Gilman A. G. (1991) J. Biol. Chem. 266, 46544659. 34. Iniguez-Lluhi J. A., Simon M. I., Robishaw J. D. and Gilman A. G. (1992)J. Biol. Chem. 267, 23409-23417. 35. Matsuda T., Takao T., Shimonishi Y., Murata M., Asano T., Yoshizawa T. and Fukada Y. (1994) J. Biol. Chem. 269, 30358-30363. 36. Sternweis P. C. (1986) J. Biol. Chem, 261,631-637. 37. Mumby S. M., Heukeroth R. O., Gordon J. I. and Gilman A. G. (1990) Proc. Natl. Acad. Sci. USA 87, 728-732. 38. Pronin A. N. and Gautam N. (1993) FEBS Lett. 328, 89-93. 39. Morishita R., Fukada Y., Kokame K., Yoshizawa T., Masuda K., Niwa M., Kato K. and Asano T. (1992) Eur. J. Biochem. 210, 1061-1069. 40. Ohguro H., Fukada Y., Takao T., Shimonishi Y., YoshizawaT. and Akino T. (1991) EMBOJ. 10, 3669-3674. 41. BigayJ., Faurobert E., Franco M. and Chabre M. (1994) Biochemsitry 33, 14081-14090. 42. Parish C. A. and Rando R. R. (1994) Biochemistry 33, 99869991. 43. Clapham D. E. and Neer E. J. (1993) Nature 365,403--406. 44. Iniguez-Lluhi J., Kleuss C. and Gilman A. G. (1993) Trends Cell. Biol. 3,230-236. 45. Kisselev O. and Gautam N. (1993) J. Biol. Chem. 268, 24519-24522. 46. Kisselev O. G., Ermolaeva M. V. and Gautam N. (1994) J. Biol. Chem. 269, 21399-21402. 47. Kisselev O., Ermolaeva M. and Gautam N. (1995) J. Biol. Chem. 270, 25356-25358. 48. Tang W.-J. and Gilman A. G. (1992) Cell 70, 869-872. 49. Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J. and Gierschik P. (1992) Nature 360, 684-686. 50. Dietrich A., Meister M., Brazil D., Camps M. and Gierschik P. (1994) Eur. J. Biochem. 219, 171-178.
ISSN 0898-6568/96 $15.00 PII S0898-6568(96)00071-X ELSEVIER
The Role of Prenylation in G-Protein Assembly and Function Joyce B. Higgins* and Patrick J. Casey DEPARTMENTS OF MOLECULAR CANCER BIOLOGY AND BIOCHEMISTRY,
DUXE UNIVERSITYMEDICALCENTER,DURHAMNC 27710-3686 USA
ABSTRACT. Heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are vital components of numerous signal transduction pathways, including sensory and hormonal response systems. G-proteins transduce signals 8om heptahelical transmembrane receptors to downstream effectors.The localizationof a G-protein to the plasma membrane, as well as its interaction with the appropriate receptor and effector, are essential for its function. In addition, the association of a G-protein's subunits to form its trimer is required for interaction with its receptor. The G-protein y subunits (G~) are subject to a set of carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and methylation. Recent advances which elucidate the contributions that the post-translational modifications of the Gv subunit have on the assembly, membrane association, and function of the G-protein trimer reveal that these modifications are required for important protein-protein, in addition to membrane-protein, interactions. Copyright ©/996 Elsevier Science Inc. CELLSmNAL8;6:433-437, 1996. KEY WORDS. G-protein, Prenylation, Isoprenylation, Subunit interaction, Processing, Famesyl, Geranylgeranyl, Methylation
INTRODUCTION Guanine nucleotide-binding regulatory proteins (G-proteins) are signal mediators in a number of pathways that involve the heptahelical family of transmembrane receptors and a variety of downstream effectors [1, 2]. G-proteins comprise three subunits, denoted G~, G~, and G~, which in the inactive state form a heterotrimer, designated G ~ . G-proteins transduce extracellular stimuli into cellular changes through a common mechanism. Association of the heterotrimer with a ligand-activated heptahelical receptor stimulates a conformational change in G~, allowing the release of guanosine diphosphate (GDP) and subsequently the binding of guanosine triphosphate (GTP). The G~ and G~ subunits dissociate from the GTP-bound G~ as a tight complex (G~). Both the free G ~ complex and the activated G~-GTP are able to act independently, synergistically, or antagonistically on specific effector molecules to alter second-messenger concentrations and thus alter specific cellular processes. The hydrolysis of the bound GTP to GDP and the release of P~ leads to inactivation of G~, re-association of the G~ with G~, and finally re-coupling of the heterotrimer to the receptor if the agonist is still present. Nearly all the players in these signalling processes are localized to the plasma membrane, either through peripheral or integral membrane association [14]. * Address all correspondence to Joyce B. Higgins, Department of Chemistry, Eastern Illinois University, Charleston, IL 61920. Received 6 January; and accepted 28 February 1996.
Although membrane association of G-proteins is essential for their biological activity, none of the three subunits contains a transmembrane-spanning domain to account for the membrane interactions. Recent discoveries have revealed that essentially all of the G proteins are instead modified by covalently attached lipid moieties [4-6]. Three types of lipids have been found covalently linked to G-protein subunits (Fig. 1). The G~ subunits are modified by myristate, a 14-carbon saturated fatty-acyl group; palmitate, a 16-carbon fatty-acyl chain; or by both groups. The myristate is linked to G~ through an amide bond to the glycine residue at the amino terminus [7]. This modification occurs co-translationally and is chemically stable to all but strong acid. Palmitate, on the other hand, is post-translationally attached to G~ through a base-labile thioester bond to the cysteine residue(s) near the amino terminus [8, 9]. Palmitoylation is a reversible modification whose lability may play a dynamic role in signal-transduction regulation [5, 10]. The G~ subunits of G-proteins contain the so-called -CaaX motif at their carboxyl terminus, and are modified by a process termed prenylation. This post-translational modification includes transfer of an isoprenoid lipid from a prenyldiphosphate substrate to the invariant cysteine of the -CaaX motif by specific protein prenyltransferases [11]. Proteins can be modified by one of two distinct isoprenoids, the 15-carbon famesyl or the 20-carbon geranylgeranyl. The G~I subunit associated with the retinal G-protein is famesylated [12, 13], as is an additional, recently discovered Gy
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well as a prenyl moiety, it has been proposed that the molecule may be involved in the trafficking of prenylated proteins to the intracellular membrane, where the proteolysis and methylation occur. The lipid modifications of the G-protein subunits not only contribute to membrane interaction of the proteins, but apparently also contribute to critical protein-protein interactions in the signalling pathways they control. This review focuses on the contributions of the G~ modifications to the assembly, membrane association, and function of trimeric G-proteins.
HETEROTRIMER FORMATION
FIGURE 1. Lipid modifications of G-proteins. The three types of lipid modifications found on G-proteins are depicted here on the basal state of the heterotrimer associated with the plasma membrane. The G~ subunit depicted contains both a thioesterlinked palmitoyl group and a myristoyl group attached via an amide bond to the N-terminal glycine. The G~ subunit contains a geranylgeranyl isoprenoid linked through a thioether bond to a cysteine residue that also contains a methylester. While all the lipids are shown inserted into the membrane bilayer, the existence of additional membrane proteins involved in the membrane association has not been ruled out.
[14], whereas the remainder of known G~ subunits are modified by the geranylgeranyl moiety [14-16]. As with essentially all -CaaX type proteins, prenylation is followed by cleavage of the last three residues of Gy by an endoprotease, and finally the carboxyl terminal prenylcysteine is methylated by a methyltransferase [11, 17]. While the first two processing events (i.e., prenylation and proteolysis) are stable modifications, the methylester bond is somewhat labile. Thus, the methylation state of prenylated proteins may play a dynamic role in the regulation of signaltransduction pathways [18, 19]. Enzymes capable of both the methylation and demethylation of prenylated proteins have been identified in retinal membranes [20]. Another potential player in G~ processing is a binding protein that has been identified in the microsomal membrane fraction, and which recognizes both farnesylated and geranylgeranylated proteins [21]. Since high-affinity binding to this site requires the last three amino acids of the -CaaX motif as
As noted earlier, G-proteins exist as trimers in the basal state, and G~ dissociates from the GTP-bound G~ subunit upon activation. The extremely stable G~ dimer is also resistant to proteolysis [22]; this property has been exploited in studies assessing G~ dimer assembly. Even after cleavage at this single site, the G~ and Oysubunits remain tightly associated without any disulfide bonds; surprisingly, the cleaved complex can still form a functional trimer with G~ [23]. The first successful reconstitution of G~y dimers was accomplished by assembling G~ and G~ proteins translated in rabbit reticulocyte lysates [24]. Further studies demonstrated that reconstitution was dependent on the system utilized for the expression of G~, but not that for G~ [24, 25]. Gy expressed and purified from systems as evolutionarily divergent as bacteria, insects, and animals are all competent for in vitro assembly [24-26]. However, while G B subunits expressed in both insect cells and rabbit reticulocyte lysates are competent for in vitro assembly [24, 25], those expressed in wheat-germ extracts or Escherichia coli are not [25, 27]. Formation of a stable dimer by co-expression of G~ and G~ in vivo also depends on the cell type; co-expression in COS cells and insect cells produces stable dimers [24, 28-31], whereas co-expression in bacteria is not suitable for G~ dimer formation [25]. The expression conditions required to produce or reconstitute stable G~y dimers indicate that an accessory protein or proteins may facilitate the assembly reaction or prevent G~ aggregation prior to assembly. The assembly of G~y dimers is most probably a cytosolic event that occurs prior to the processing of the G~ subunit [25, 26]. Substitution of Cys6s in G~2 (the site of isoprenoid addition) with Ser prevents the prenylation of his polypeptide, and its co-expression with G~ results in production of a cytosolic G~ complex [29, 32]. These studies demonstrated that the assembly of G~ dimers is not dependent on prenylation of G~. Reconstitution of G~ dimers utilizing G~ expressed in Sf9 cells and G~ purified from E. coli has provided additional information in this regard. Unmodified and prenylated G~ subunits can assemble with G~, but the unmodified form is more efficient [26]. These data confirm that prenylation is not required for G~ assembly. On the other hand, the truncated-prenylated G~ protein (i.e., G~ that is missing the "-aaX" residues of the -CaaX motif) does not assemble with G~, suggesting that assembly must occur before proteolysis [25]. The fully processed G~ subunit is
Prenylation in G-protein Assembly and Function
435
LaaLi FIGURE 2. Model for assembly and processing of Gpv. In this model, the G~ and Gv proteins synthesized in the cytosol associateto form stable G~v dimers. The soluble Gp~ complex thus formed serves as a substrate for enzymatic prenylation of the Gv polypeptide. The prenylated dimer then translocates to the microsomal membrane through associating with a specific binding protein, whereupon the processing is completed by actions of an endoprotease that cleaves the three C-terminal residues, followed by methylation of the exposed prenylcysteine. The mature protein is then targeted to the plasma membrane by mechanisms even less clear.
CaaX
I
even less efficient in assembly with G~ than the Gv subunit that is only prenylated, and G~ complexes containing an unmodified G~ serve as excellent substrates for the protein prenyltransferase, demonstrating that prenylation can occur after complex assembly [26]. Overexpression of Gy subunits in COS cells results in an accumulation of Gv-proteins on an intracellular membrane, perhaps because the proteolysis or methylation of the prenylated proteins, which occurs on intracellular membranes, becomes rate-limiting, and overexpression of G~ and Oysubunits in the same cell shows accumulation of both subunits at the intracellular membrane [29]. Taken together, these results indicate that processing of Gy most probably occurs after assembly of the G~s complex. The association of G~ and G~ into the trimeric G ~ is necessary not only to attenuate signalling of both the activated G~ and the free G~v complex, but also for recognition of the G~ subunit by the receptor and for enhanced membrane association (see below). Myristoylation of G~ subunits enhances their interaction with G~ [33]. Although prenylation of G~ is not required for the assembly of G~ complexes, the G~ complex does require a prenylated Gv for high-affinity interaction with G~ [25, 34]. Whether the requirements for lipid modifications of both G~ and G, for
Protein Prenyltransferasa •.W.,J.~P ~ ~
NH3+
high-affinity trimer formation reflect an interaction of these lipids in the assembled species is not yet clear. Methylation of the carboxyl-terminal prenylcysteine in Gyl increases the interaction of G,~ with O131y] by 2-fold over the unmethylated G~v isoform [19, 35]. In addition, the Gt~G~ interaction can be inhibited by peptides corresponding to the carboxyl terminus of Gvl, but only if the peptide is farnesylated. Substituting geranylgeranyl for the famesyl group on the peptide increases the inhibition by 2-fold, whereas methylation of the farnesylated peptide increases the inhibition by 3-fold [35]. These data suggest that the degree of hydrophobicity of the G~I subunit plays a significant role in these subunit interactions. Thus, results with a wide variety of experimental approaches indicate that prenylation and subsequent processing of G~, in addition to the acyl modifications of G~, contribute to the stability of the G~O~y interaction. MEMBRANE ASSOCIATION The localization of the G-proteins to the intracellular surface of the plasma membrane increases their effective concentrations and thus facilitates their interactions with both
436 receptors and effectors. Palmitoylation, myristoylation, and Glay interaction all contribute to the membrane association of G~ [4-6]. The contribution of G~ was first demonstrated through the use of reconstitution studies. Efficient association of bovine brain G,~ with phospholipid vesicles was found to require the presence of G~, on the vesicles [36]. This is not an all-or-none event, however, since G~ subunits over-expressed in COS cells are almost exclusively found in the membrane fraction even when the total amount of G~ is in excess of the G~y concentration [37]. It seems likely that the presence of membrane-associated G~ in the cells is necessary to target O~ to the membrane, but is not essential to keep the G~ associated with the plasma membrane once it has encountered the lipid bilayer. A variety of approaches have been used to demonstrate that the membrane association of the G~s complex is predominantly the consequence of the post-translational modification of the Gy subunit. Prenylation of the G~ protein promotes the membrane association of both G~ monomers and G~ dimers [28, 29]. Co-expression of G~ and Gs increases the fraction of Gy that is prenylated and subsequently membrane-associated [30, 38], presumably through an increased stability of G~ when it has assembled with G~. The localization of the G~ complex in cells can be altered by treating the cells with the drug compactin. Compactin is an inhibitor of hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway, and treatment of cells with the drug is a technique commonly used to prevent isoprenoid synthesis and thus protein prenylation. Following treatment of cells with compactin, a fraction of the G~ complexes became cytoplasmic, while the bulk of the G~ remained membrane-associated. Membrane association of G~ could be restored by supplementing compactin-treated cells with exogenous mevalonate, the product of HMG-CoA reductase [29]. Additionally, as noted above, the C68S G~ mutant has a cytoplasmic distribution in Sf9 cells, and its co-expression with G~ results in production of a cytosolic G~ complex [29, 32]. These results demonstrate that prenylation of Gy is essential for membrane association of the G~ complex. The consequences of the proteolysis and methylation steps in G~ processing with regard to membrane association of the G~ complex have been more difficult to address, since these enzymatic steps occur quite rapidly after prenylation. While G~ complexes isolated from a number of different tissue sources apparently contain only methylated G~ [39], retinal G~ complexes are found as a mixture of complexes containing both unmethylated and methylated G~ [40, 41]. Separation of these two forms revealed that the rnethylated form has a higher affinity for the retinal membranes than the unmethylated form. Moreover, the presence of G~ significantly enhanced the association of the methylated G~ to the membrane, but only slightly increased the membrane association of the unmethylated form [40]. Similar results have been obtained using G~s complexes that were demethylated by esterase treatment after purification [42]. These data indicate that the methyl-
J. B, Higgins and P. J. Casey ation state not only increases the affinity of G~ for membranes but also its affinity for G~, and also that membrane binding of G~ is influenced by G~. Additional information on interaction of the G-protein subunits with the lipid bilayer has come from reconstitution studies using phospholipid vesicles of defined composition, which have provided evidence that, in addition to the lipid modifications, charged amino acid domains also contribute to the membrane association of both the G~ complex and G~ subunit [35]. These data have also been interpreted as suggesting that the domains responsible for the membrane attachment of the individual subunit complexes (i.e., G~, G~-GTP) are different from that of the heterotrimer.
G PROTEIN FUNCTION After years of languishing in obscurity, the G~ complex has finally received its rightful share of attention for active roles in G-protein function. Since 1987, numerous protein targets that are regulated by G~ have been identified [43, 44]. The elucidation of multiple activities for the G~ complex has transformed the classical view of G-protein signalling and has emphasized the importance of understanding the mechanisms of G~ interactions. The recognition of the heterotrimer by the receptor is one of the first steps in the signalling pathway. G-proteincoupled receptors appear to be more discriminating than G~ subunits with regard to their interaction with specific G~ dimers. While the G,~ subunit will bind G~ complexes composed of G~I and a number of G~ isotypes, coupling of the transducin heterotrimer to its receptor, rhodopsin, is more efficient with the trimer containing the Gs~ isotype [45]. Metarhodopsin II, the receptor conformation that is stabilized by the binding of transducin, requires prenylated G~, and preferentially binds transducin containing the methylated Gyl subunit [19, 40]. In fact, a farnesylated peptide corresponding to the C-terminus Gyl can uncouple the transducin trimer from the receptor and stabilize metarhodopsin II [46], and the farnesyl group itself appears to be a critical determinant in this interaction [47]. The preference of both the receptors and the G~ subunits for the processed cysteine on G~s indicates that the modifications of the Gs subunit contribute to protein-protein interactions essential to G-protein function. As noted above, the list of effector molecules that are regulated by G~ is growing, and many of these effectors can be regulated not only by G~ but by Go as well. A particularly well-studied example is the regulation of adenylyl cyclase. Some isotypes of this enzyme are inhibited by the G~ complex, while others are synergistically stimulated by G~ in conjunction with activation by G,~ [48]. The effects of the G~ complex on both classes of adenylyl cyclase require processed G~ [34]. Another effector that has been extensively studied is the 132 isotype of phospholipase C (PLCI3/); this protein is independently activated by both the G~ and G~ subunits [34, 49]. Prenylation of the G~ subunit is necessary for the activation of PLC[~2 by G~ in intact cells and in re-
Prenylation in G-protein Assembly and Function constituted phospholipid vesicle systems [50], and the effect is enhanced by methylation of the prenylcysteine [18]. Although the precise contributions of G~ processing to effectorG~v interactions have not been fully analyzed, particularly the respective roles of enhanced membrane association versus a direct influence on protein-protein interactions, current observations indicate that the processing of the G~ subunit in the O~y complex is essential for G ~ regulation of most effector molecules. CONCLUSIONS Signal-transduction pathways mediated by trimeric G-proteins are numerous, and the interactions of the G-protein subunits with the components of these systems are critical determinants of their activity and specificity. The mechanism by which the G-protein subunits interact with each other, with other signalling molecules, and with the membrane surface are of definite import. Although the contributions of the covalent modifications of the G~ and G~ subunits are significant, they are certainly not the sole determinants of these subunits' effects. Nonetheless, an increased awareness of the importance of lipid modifications in these processes should lead to a more detailed description of the molecular events involved in these signalling processes. Dr. Casey is an establishedinvestigatorof the American Heart Association.
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