ELLIOTT M. Ross PROTEIN MODIFICATION

Palmitoylation in G-protein signaling pathwaysThe reversible palmitoylation of G proteins and their receptors isinvolved both in receptor desensitization and in association and

disassociation of Gex subunits with the plasma membrane.

Some say that geography is destiny. Cell biologists feelthis way, and biochemists are sometimes forced to agree.Exactly where a signaling protein resides in a cell canmarkedly influence what it does, how well it does it andto whom it does it. In signal transduction pathways, con-trolled subcellular localization of receptors, G proteinsand effectors influences both the fidelity of signaling -who talks to whom - and the long-term control ofsignaling through receptor endocytosis and down-regula-tion. Over the past few years, the reversible palmitoy-lation of both G proteins and G-protein-coupled recep-tors has been recognized as one way of regulating theirlocalization and activity.

Both G protein aL subunits and G-protein-coupled recep-tors are palmitoylated through thioester linkages tocysteine residues. The receptors, which are intrinsicmembrane proteins based on a bundle of seven hydro-phobic, membrane-spanning helices, are palmitoylated intheir carboxy-terminal cytoplasmic domains, about 12residues past the end of the seventh helix. Some receptorsterminate just after this residue, but others contain exten-sive carboxy-terminal domains of 150 amino-acid residuesor more.

G protein x subunits, which bind GTP and which inter-act directly with both receptors and effectors, are palmi-toylated near their amino termini, usually at residue 3.The most typical mature amino-terminal structure is anN-myristoylated glycine followed by an S-palmitoylatedcysteine. These hydrophobic modifications of Got sub-units are in addition to the prenylation, usually by ageranylgeranyl thioether, of Gy subunits on the carboxy-terminal cysteine methyl ester.

In the case of the G proteins, which do not have notablyhydrophobic amino-acid sequences and which bind atthe inner surface of the plasma membrane, palmitoylationand other alkylations increase their overall hydropho-bicity and promote their attachment to the membrane.For the already hydrophobic receptors, the differentialhydrophobicity contributed by a palmitoyl group is triv-ial. In either case, why has biology chosen alkylation as away of increasing hydrophobicity rather than the perma-nent addition of a few more leucine or valine residues?There are at least two answers.

First, palmitoylation may enhance binding to particularproteins rather than just enhancing membrane attach-ment. Such recognition of specific cellular structures can

change the location of receptors or G proteins in cells.The reversibility of the palmitoylation, triggered directlyor indirectly by an agonist, raises the even more enticingprospect that the cell can regulate both the relocaliza-tion of signaling proteins and their associations withother proteins.

Regulated turnover of receptor palmitoylationEver since the palmitoylation of rhodopsin was firstobserved in 1988 [1], a small but steady series of papershas described palmitoylation of other G-protein-coupledreceptors; nearly all the receptors contain the palmitoy-latable cysteine residue after the seventh span. But muta-tion of this cysteine- generally has subtle, if any, effects.An engineered rhodopsin mutant with no cytoplasmiccysteine residues is functionally wild type when measuredin an in vitro reconstituted assay [2]. Similarly, cysteinemissense mutants of 132-adrenergic [3], D 1 dopaminergic[4] and eo2A-adrenergic [5] receptors, which do notbecome palmitoylated, have all been reported to displayunaltered signaling phenotypes when expressed (at fairlyhigh levels) in cultured cells. In another study, a similarcysteine mutation in the 132-adrenergic receptor causedonly subtle changes in G-protein coupling [6,7].

Two observations by Bouvier and co-workers [3,4] madereceptor palmitoylation more intriguing by linking it toreceptor desensitization, which occurs upon the contin-ued exposure of cells to agonists. Desensitization andsubsequent resensitization of G-protein-coupled recep-tors are the result of a complex set of processes. Theyinclude receptor phosphorylation by at least two proteinkinases, decreased coupling to G protein (probablycaused both by phosphorylation and by the binding ofinhibitory proteins known as arrestins), receptor endo-cytosis and degradation, and (frequently) a decrease innew receptor synthesis.

How does palmitoylation fit in with these processes?Mouillac et al. [3] reported that continued exposure ofcells to agonist increases the slow, in vivo labeling of32-adrenergic receptors with [3H]palmitic acid, an obser-

vation later confirmed for D 1 receptors [4]. Of evengreater interest, a non-palmitoylated mutant receptor(Cys341Gly) displayed several features of desensitizationwithout exposure to agonist. The mutant receptor wasactive in signaling, but a little less so than the wild type.Unlike the wild-type receptor, however, the mutant wasnot much uncoupled by continued agonist exposure. Themutant receptor was also more highly phosphorylated

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than the wild type in the absence of agonist, but was notphosphorylated much further when agonist was added.Similar observations were made for an analogous mutantD 1 receptor [4].

When these phenomena are put together and some tech-nical inadequacies overlooked, they make a story thatlinks turnover of palmitoyl groups to receptor desen-sitization. The simplest version of the story is that un-stimulated receptor in the plasma membrane is largelypalmitoylated. When stimulated by agonist, the receptornot only activates G protein, but also becomes a bettersubstrate for the enzyme palmitoyl-protein thioesterase,which removes palmitoyl groups [8]. Depalmitoylationtriggers receptor phosphorylation, consequent uncoup-ling and (perhaps) later endocytosis. In this scheme, theobserved stimulation of labeling with [3 H]palmitate inresponse to continued exposure to agonist reflects somepart of the resensitization pathway, perhaps that involved.in movement of receptors into or out of endosomes [9].More detailed speculations can be left to the imaginationof the reader.

Now for the hard part. So far, it has not been possibleto measure receptor palmitoylation. Labeling with[3H]palmitate is expensive, inefficient and hard to detect- two-week exposure of fluorographs of gels ofimmunoprecipitates or affinity-purified receptor is thenorm. Further, labeling with [3H]palmitate may reflectincreased steady-state palmitoylation, unchanged netpalmitoylation combined with rapid turnover, or even anet decrease in palmitoylation if thioesterase activity ishigh. So far, there are no data that speak (even qualita-tively) to the loss of palmitate from prelabeled receptor.Second, it is unclear which population of receptor -palmitoylated or unpalmitoylated - is under observationin assays of signaling, phosphorylation or endocytosis.Last, some receptors lack the palmitoylated cysteine yetundergo the cycle of uncoupling, endocytosis and recov-ery. Thus, although receptor palmitoylation certainlyseems to be involved in modulating sensitivity to agon-ists, and in very interesting ways, its control and mecha-nism of action remain open questions for the G-proteinsignaling community.

Ga subunit palmitoylation and depalmitoylationLike the receptors that regulate them, G protein subunits also undergo a cycle of depalmitoylation andpalmitoylation. G subunits are palmitoylated on aconserved cysteine residue, usually found at position 3[10,11], and palmitoylation enhances Got binding tomembranes. A non-palmitoylated mutant of Goot isfound primarily in the cytosol, and the analogous Cys3mutants of Gsot and Gqa, the wild-type forms of whichbind more tightly to membranes than does Got, arefound in cytosol fractions at least to some extent [12,13].This diminished membrane binding may in fact com-pletely account for the diminished signaling activitiesnoted in some of these mutants [13], because non-palmi-toylated Ga subunits are active in vitro. G, Goa and

Gqao are all largely depalmitoylated during purification, asassessed by their abilities to form disulfides either withmastoparans (short receptor-mimetic peptides [14]) orwith another cysteine residue near the carboxyl terminus(T. Higashijima and myself, unpublished data). All, how-ever, remain responsive to receptors and purified Gsoaand Gqoa efficiently activate their respective effectorproteins, adenylyl cyclase and phospholipase C-P.

Like receptors, Ga subunits undergo accelerateddepalmitoylation and repalmitoylation when they areactivated within the cell during signal transduction. Theeffects of activation on palmitate turnover on Gat aresimilar whether activation is by receptors, or is the resultof covalent modification by cholera toxin (for Gs) or of amutation that increases the rate of activation by cellularGTP [12,15,16]. In the most thorough study to date,Wedegaertner and Bourne [16] have clarified the linkagebetween the activation and palmitoylation cycles, andhave indirectly estimated the stoichiometry of palmitateturnover. First, they found that activation increased therate of depalmitoylation of Gsot that had been prelabeledwith [3H]palmitate. Activation promoted completeremoval of [3 H]palmitoyl groups. They argue convin-cingly that the kinetics of labeling and the subcellularlocalization of G a are consistent with activation-induceddepalmitoylation of membrane-bound Gsa. Depalmi-toylation allowed the Got to leave the plasma membrane,and repalmitoylation occurred in the cytoplasm. Becauseboth they and Degtyarev et al. [15] had found that amutation that inhibits dissociation of Gao from GJy alsoinhibits the palmitate cycle, Wedegaertner and Bourneinferred that dissociation of activated Ga from Gyprecedes hydrolysis of the palmitoyl group and thatdepalmitoylation is the decisive event in releasing Gafrom the membrane. This idea is supported by the obser-vation that depalmitoylation of detergent-solubilized Gsoawas inhibited by GJ3y.

The conclusion of this work, assuming that it is con-firmed, is that activation-promoted depalmitoylation ofGoa subunits causes their slow transfer to the cytosol.Does this mean that the Got subunits activate cytosolictargets while there? Probably not. Although G proteinsdeactivate slowly, all but the slowest hydrolyze theirbound GTP in less than 3 minutes at 37 OC; Gs and Giare much faster than this. Thus, by the time muchdepalmitoylated Ga gets to the cytosol, it is in the inac-tive, GDP-bound state. The predominant cytosolicspecies would thus be inactive. Depalmitoylation is moreprobably involved in long-term regulation of the signal-ing activity of Ga subunits, perhaps by altering theirassociations with other important proteins. Results withthe Cys 3 mutants are consistent with this idea.

True solubilization of Ga subunits by removal of apalmitoyl anchor may also be an oversimplification. Asargued by Resh [17], the hydrophobicity of a palmitoylgroup is insufficient to anchor a protein to a bilayer. Theobserved release might indicate that palmitoylation is

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important for binding to another membrane protein,Gpy perhaps. Alternatively, the observed release mayonly reflect the massive dilution of cytosol that occurswhen cells are homogenized. In cells, depalmitoylatedGa may never leave the membrane but only redistributelaterally. The questions that remain are where thedepalmitoylated Ga goes, what it does when it gets thereand how it gets palmitoylated again and returned to thesignaling switchboard.

References1. Ovchinikov YA, Abdulaev NG, Bogachuk AS: Two adjacent cys-

teine residues in the C-terminal cytoplasmic fragment of bovinerhodopsin are palmitylated. FEBS Lett 1988, 230:1 5.

2. Karnik SS, Sakmar TP, Chen H-B, Khorana HG: Cysteine residues110 and 187 are essential for the formation of correct structure inbovine rhodopsin. Proc Natl Acad Sci USA 1988, 85:8459-8463.

3. Mouillac B, Caron M, Bonin H, Dennis M, Bouvier M: Agonist-modulated palmitoylation of 32-adrenergic receptor in Sf9 cells.J BiolChem 1992, 267:21733-21737.

4. Ng GY, Mouillac B, George SR, Caron M, Dennis M, Bouvier M,O'Dowd BF: Desensitization, phosphorylation and palmitoylationof the human dopamine D, receptor. Eur Pharmacol 1994,267:7-19.

5. Kennedy ME, Limbird LE: Mutations of the ta2A-adrenergic recep-tor that eliminate detectable palmitoylation do not perturb recep-tor-G-protein coupling. I Biol Chem 1993, 268:8003-8011.

6. O'Dowd BF, Hnatowich M, Caron MG, Lefkowitz RI, Bouvier M:Palmitoylation of the human ,32-adrenergic receptor. Mutationof CYS341 in the carboxyl tail leads to an uncoupled non-palymitoylated form of the receptor. Biol Chem 1989, 264:7564-7569.

7. Moffet S, Mouillac B, Bonin H, Bouvier M: Altered phosphorylationand desensitization patterns of a human 32 -adrenergic receptorlacking the palmitoylated Cys341. EMBO 1 1993, 12:349- 356.

8. Camp LA, Verkruyse LA, Afendis SJ, Slaughter CA, Hofmann SL:Molecular cloning and expression of palmitoyl-protein thioester-ase. J Biol Chem 1994, 269:23212-23219.

9. Yu SS, Lefkowitz RJ, Hausdorff WP: -Adrenergic receptor seques-tration. A potential mechanism of receptor resensitization. J BiolChem 1993, 268:337-341.

10. Parenti M, Vigano MA, Newman CMH, Milligan G, Magee Al: Anovel N-terminal motif for palmitoylation of G-protein a subunits.J Biochem 1993, 291:349 353.

11. Linder ME, Middleton P, Hepler JR, Taussig R, Gilman AG, MumbySM: Lipid modifications of G proteins: a subunits arepalmitoylated. Proc Natl Acad Sci USA 1993, 90:3675-3679.

12. Mumby SM, Kleuss C, Gilman AG: Receptor regulation of G-pro-tein palmitoylation. Proc Natl Acad Sci USA 1994, 91:2800-2804.

13. Wedegaertner PB, Chu DH, Wilson PT, Levis MJ, Bourne HR:Palmitoylation is required for signaling functions and membraneattachment of Gqa and G,a. J Biol Chem 1993, 268:25001-25008.

14. Higashijima T, Ross EM: Mapping of the mastoparan-binding site onG proteins: cross-linking of [1251-Tyr3,Cys11]mastoparan to Go.J Biol Chem 1991, 266:12655-12661.

15. Degtyarev MY, Spiegel AM, Jones TLZ: Increased palmitoylation ofthe Gs protein a subunit after activation by the 13-adrenergic recep-tor or cholera toxin. Biol Chem 1993, 268:23769-23772.

16. Wedegaertner PB, Bourne HR: Activation and depalmitoylation ofGso. Cell 1994, 77:1063-1070.

17. Resh MD: Myristylation and palmitylation of Src family members:the fats of the matter. Cell 1994, 76:411-413.

Elliott M. Ross, Department of Pharmacology, Univer-sity of Texas Southwestern Medical Center, 5323 HarryHines Boulevard, Dallas, Texas 75235-9041, USA.

THE APRIL 1995 ISSUE (VOL. 7, NO. 2) OFCURRENT OPINION IN CELL BIOLOGY

will include the following reviews, edited by Axel Ullrich and Mike Simons, on Cell Regulation:

Lysophosphatidic acid signalling by W. MoolenaarMolecular control of life and death by J. Yan

InsP3 receptor mediated spatiotemporal Ca2+ signalling by S. MiyazakiNeurotrophic factors and their receptors by M. Barbacid

The IL-2 signaling; recruitment and activation of multiple protein tyrosine kinases by theIL-2 receptor components by T. Taniguchi and Y. Minami

The GRB2/SHC/SOS/ras signaling pathway by J. SchlessingerRole of src family members in cellular signal transduction by S. Courtneidge

Significance of Ptdlns(4,5)P 2 hydrolysis and regulation of phospholipase C isozymesby S. Goo Rhee

Abscisic acid signaling in plants byJ. GiraudatTwo-component signaling systems by M. Simon

T and B cell receptor signal transduction by A. DeFrancoRole of SH2 and SH3 domains in signalling in tyrosine kinases, concentrating on

new insights gained from 3D structures byJ. KuriyanUbiquitin, proteasomes, and the regulation of intracellular protein degradation by M. Hochstrasser

The auxin signal in plants by P. MillnerThe proliferation of MAP kinase signaling pathways in yeast by D.E. Levin and B. Errede