©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fatty Acylation of
EFFECTS OF PALMITOYLATION AND MYRISTOYLATION ON SIGNALING (*)

Paul T. Wilson (1) (4), Henry R. Bourne (2) (3) (5)(§)

From the (1) Departments of Psychiatry, (2) Pharmacology, and (3) Medicine, the (4) Center for Biology and Psychiatry and the (5) Cardiovascular Research Institute, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

As the first step in an investigation of roles played by fatty acylation of G protein chains in membrane targeting and signal transmission, we inserted monoclonal antibody epitopes, hemagglutinin (HA) or Glu-Glu (EE), at two internal sites in three subunits. At site I, only HA-tagged and functioned normally. , , and subunits tagged at site II with the EE epitope showed normal expression, membrane localization, and signaling activity. Using epitope-tagged , we investigated effects of mutations in sites for fatty acylation. Mutational substitution of Ala for Gly(G2A) prevented incorporation of myristate and decreased but did not abolish incorporation of palmitate. Substitution of Ala for Cys(C3A) prevented incorporation of palmitate but had no effect on incorporation of myristate. Substitution of Ala for both Glyand Cys(G2AC3A) prevented incorporation of both myristate and palmitate. All three mutations substantially disrupted association of with the particulate fraction. G-mediated inhibition of adenylyl cyclase, triggered by activation of the D2-dopamine receptor, was, respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A). Constitutive inhibition of adenylyl cyclase by was unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced (C3A). A nonacylated, mutationally activated mutant inhibited adenylyl cyclase, although less potently than normally acylated, mutationally activated . From these findings we conclude: ( a) fatty acylations of increase its association with membranes; ( b) myristoylation is not required for palmitoylation of or for its productive interactions with adenylyl cyclase; ( c) palmitoylation is not required for, but may instead inhibit, signaling by .


INTRODUCTION

Hetereotrimeric () G proteins transduce signals from cell surface receptors to a variety of effectors, including adenylyl cyclase, ion channels, and phospholipase C. Covalent attachment of myristate and/or palmitate to G protein subunits greatly facilitates and is in some cases essential for their association with the cytoplasmic surface of the plasma membrane and for normal signaling function. These characteristics make G protein subunits attractive models for investigating roles played by distinct fatty acylations in membrane association and protein function.

Myristoylation of subunits involves covalent addition of myristate to a Glyresidue via an amide linkage, and takes place during or immediately after translation. This modification is usually irreversible, but not always (1) and therefore an unlikely target for regulation. Myristoylation is confined to members of the family, where it serves dual roles promoting membrane attachment and increasing affinity of protein-protein interactions. Mutations that abolish myristoylation substantially impair attachment of , , and to membranes (2, 3, 4) . Myristoylation promotes membrane attachment of and , at least in part, by increasing their affinity for membrane bound subunits (5, 6) . Productive interaction of with adenylyl cyclase requires myristoylation (7) .

Excepting , all subunits thus far examined are palmitoylated (8, 9, 10) . In contrast to myristoylation, palmitoylation is a dynamic, reversible modification involving post-translational attachment of palmitate to a Cys residue by a thioester linkage. For and , which are modified solely by palmitate, this modification plays a critical role: mutations that prevent palmitoylation markedly impair membrane association (8, 9, 11, 12) and signaling functions (12) of and . Moreover, palmitoylation is regulated (13, 14, 15) . Following activation of in intact cells, palmitate is removed, and depalmitoylated is found in the soluble compartment, suggesting that activation-dependent depalmitoylation may serve to decrease the amount of available for subsequent interactions with receptors and effectors (15) . The function and regulation of palmitate in subunits that are modified by both palmitate and myristate awaits elucidation.

Here we report experiments designed to examine the effects of palmitoylation and myristoylation on membrane attachment and signaling function of . A member of the family, is expressed primarily in brain, adrenal gland, and platelets (16) . Like , can mediate hormonal inhibition of adenylyl cyclase (17) and is both palmitoylated and myristoylated (2, 4) . Unlike , is not uncoupled from receptor activation by the action of pertussis toxin (PTX),() because it lacks the Cys residue modified by PTX. Consequently, signals mediated by transfected can be assayed specifically and conveniently in cells treated with PTX. For this reason, we chose as an early target for a molecular genetic investigation of the roles of attached palmitate and myristate in modifying signaling by G proteins.

Because the molecular genetic approach to this problem requires easy detection and specific quantitative immunoprecipitation of mutant subunits, we began by extending the epitope tagging strategy, first employed with (18) . Here we report results of a search for a common site in G protein subunits suitable for epitope tagging. Success of this search, with respect to and , as well as , will facilitate investigation of other G protein subunits by strategies that utilize mutation, immunoprecipitation, and immunofluorescence.


EXPERIMENTAL PROCEDURES

Materials

CHO K1 and HEK293 cells were obtained from the American Type Culture Collection. cDNA constructs obtained as described in Ref. 12. Receptor agonists were from Research Biochemicals Inc. (quinpirole, UK-14304 as part of National Institute of Mental Health Chemical Synthesis Program Contract 278-90-0007[BS]), the National Pituitary Agency (human chorionic gonadotrophin), or Sigma (isoproterenol). Antisera were the generous gifts of S. Mumby and A. Gilman (G) and Paul Sternweiss (). [2-H]Inositol and [2-H]adenine were obtained from Amersham Corp. [9,10-H]Palmitic acid and [9,10-H]myristic acid were from DuPont NEN.

Plasmid Construction

Single stranded DNA corresponding to the antisense strand was prepared from pcDNA1 containing subunit constructs. Hemagglutinin (HA) or Glu-Glu (EE) epitope sequences were introduced by the site-directed mutagenesis protocol of Kunkel et al. (19) . Sense strand oligonucleotides comprising the coding sequence for either epitope flanked on both sides by 24 bases of coding sequence of the appropriate subunit were used as primers. Coding sequences were 5`- gacgtccccgactacgcg for the 6-amino acid HA epitope DVPDYA or 5`- tacgacgtccccgactacgcgtca for the 8-amino acid HA epitope YDVPDYAS. Coding sequences for the EE epitope were 5`- gagtacatgccgatggag for the sequence EYMPME, 5`- gagtacatgccgatcgag for the sequence EYMPTE, or 5`- gcggagtacatgccgatctgag for the sequence AEYMPTE used in EE-N. The G2A, C3A, and G2AC3A mutations were constructed in a similar fashion using the antisense strand of pCDNA1 containing EE-II. Mutagenesis was verified by DNA sequencing. For transient expression in S49 cyccells, constructs were subcloned into the expression vector pSR296 (20) by blunt-ended ligation at the unique PstI site.

Transient Transfection Protocols

HEK293 cells were propagated in minimal essential medium (MEM) containing 10% fetal bovine serum. Plasmids were transfected using Transfectam lipofectant (Promega). A DNA-lipofectant mixture (ratio, 1 µg of DNA to 2 µl of Transfectam) was added to a 60-mm dish of subconfluent cells bathed in 0.5 ml of serum-free MEM. After 2 h incubation, the medium was replaced with 4 ml of MEM, 10% fetal bovine serum. For assaying signaling activity of or constructs, 1 µg of R plasmid was cotransfected with 3 µg of or plasmid.

CHO K1 cells were propagated in MEM containing 10% fetal bovine serum. Initially, CHO K1 cells were transfected exactly as described for HEK293 cells except that MEM was used and the ratio of Transfectam to DNA was 4 µl/1 µg. In later experiments plasmids were transfected using the adenovirus-mediated transfection protocol (21) . Plasmid DNAs were placed into 2.5 ml of bicarbonate-free, serum-free DME H16 medium supplemented with 20 m M HEPES, nonessential amino acids, and 80 µg/ml DEAE. A 1:4 to 1:8 dilution of adenovirus stock was added to the tube and mixed. The DNA-adenovirus solution was added to a 60-mm dish of subconfluent cells. Following a 2-h incubation at 37 °C, the cells were shocked for 2 min with 10% MeSO in phosphate-buffered saline and returned to bicarbonate-free DME H16 supplemented with 20 m M HEPES, nonessential amino acids, and 10% fetal bovine serum. For assaying signaling activity of constructs, 1 µg of LHR plasmid and 2 µg of DR plasmid were cotransfected with 0-4 µg of plasmid.

S49 cyccells were propagated in DME H21 medium supplemented with 10% heat-inactivated horse serum. For transfection, cells in logarithmic growth were pelleted, washed once, and resuspended in serum-free DME H21 at a concentration of 2 10cells/ml. 225 µl of cells and 25 µl of 10 m M Tris, 0.1 m M EDTA, pH 8.0, containing 10 µg of plasmid DNA were mixed in an electroporation chamber. Electroporation was done in a Cell-Porator (BRL) at settings of 330 microfarads and 225 V. Cells were immediately transferred to 60-mm dishes containing 4 ml of DME H21 with 10% horse serum.

cAMP Assay

HEK293 or CHO K1 cells, 24 h after transfection, were reseeded into 6 wells of a 24-well plate and [H]adenine (2 µCi/ml for HEK293, 6 µCi/ml for CHO K1) was added. For assays involving constructs, pertussis toxin (200 ng/ml) was also included. 24 h later, wells were washed once with 0.5 ml of serum-free, bicarbonate-free DME H16 medium containing 20 m M HEPES (assay medium), and then incubated with 0.5 ml of assay medium containing 1 m M isobutylmethylxanthine with or without the appropriate receptor agonists. After 30 min incubation at 37 °C, the cells were lysed in 0.5 ml of 5% trichloroacetic acid containing 1 m M each cAMP and ATP. [H]cAMP and [H]ATP were separated on Dowex and alumina columns as described (22) . S49 cyccells, 24 h after transfection, were labeled with 6 µCi/ml [H]adenine for 24 h. Cells were pelleted and resuspended in 2.8 ml of assay medium. 450-µl aliquots were added to 6 wells of a 24-well plate. 50 µl of assay medium containing 10 m M isobutylmethylxanthine with or without 10 times concentration (10 M) of isoproterenol was added. During the 30-min incubation at 37 °C, the S49 cyccells adhered to the wells. Reactions were terminated with 5% trichloroacetic acid as described above. The remainder of the assay was identical to that described for HEK293 cells.

Inositol Phosphate Assay

HEK293 cells, 24 h after transfection, were reseeded into 6 wells of a 24-well plate and [H]inositol (2 µCi/ml) was added. 24 h later, wells were washed once with 0.5 ml of assay medium and then incubated with 0.5 ml of assay medium containing 10 m M LiCl with or without the receptor agonist, UK14304. After 30 min incubation at 37 °C, the cells were lysed in 0.75 ml of 20 m M formic acid. [H]Inositol phosphate and [H]inositol were separated on Dowex columns as described (22) .

Metabolic Labeling

100-mm plates of CHO K1 cells, 24 h after transfection, were reseeded into two 60-mm plates. 24 h later, the cells were washed twice with serum-free MEM and then incubated for 2 h with 1 ml of serum-free MEM containing 0.75 mCi of either [H]myristic acid or [H]palmitic acid. Cells were washed once with ice-cold phosphate-buffered saline and lysed on ice for 60 min with 330 µl of 40 m M TrisHCl, pH 8.0, containing 1 m M EGTA, 2 m M MgCl, 1% sodium cholate, and 2 µg/ml aprotinin. Lysates were transferred to Eppendorf tubes on ice and spun in a microcentrifuge at 4 °C at 3500 rpm for 3 min. Soluble material was transferred to a new Eppendorf tube on ice. Triton X-100 and SDS were added to final concentrations of 1 and 0.5%, respectively, in a total volume of 400 µl. 30 µl of EE mAb coupled to protein G-Sepharose beads (15 µl of packed beads) was added to each tube and the tubes tumbled for 1 h at 4 °C. The beads were pelleted by microcentrifugation and quickly washed with 500 µl of ice-cold phosphate-buffered saline containing 1% sodium cholate, 1% Triton X-100, and 0.5% SDS. Beads were resuspended in 100 µl of SDS-PAGE sample buffer containing 10 m M dithiothreitol and heated to 65 °C for 3 min. 30-µl aliquots were resolved on two separate 14% SDS-PA gels (18) . Gels were fixed by three 15-min washes in 50% MeOH, 10% acetic acid. After soaking for 15 min in 1 M TrisHCl, pH 7.0, one gel was transferred to a fresh solution of 1 M TrisHCl, pH 7.0, and the other to a fresh solution of 1 M hydroxylamine, pH 7.0, for 12 h. Gels were then washed twice in 50% MeOH, 10% acetic acid and processed for fluorography with Amplify (Amersham Corp.) according to the manufacturer's instructions. An additional 30-µl aliquot was resolved by SDS-PAGE and probed with biotinylated-EE mAb by Western blotting.

Cell Fractionation

100-mm plates of CHO K1 cells, 48 h after transfection, were washed with ice-cold phosphate-buffered saline. 300 µl of 40 m M TrisHCl, pH 8.0, containing 1 m M EGTA, 2 m M MgCl, and 2 µg/ml aprotinin was added to each plate on ice. Cells were scraped off the plates with a rubber policemen and transferred to Eppendorf tubes on ice. Cells were disrupted by 10 passages through a 27-gauge needle. Unlysed cells and nuclei were removed by microcentrifugation at 3,500 rpm for 5 min at 4 °C. Supernatants were separated into soluble and particulate fractions by centrifugation for 30 min at 150,000 g. Particulate fractions were resuspended into equivalent volumes of lysis buffer. Samples were analyzed by 14% SDS-PAGE followed by Western blotting.

Miscellaneous Immunological Techniques

EE mAb was obtained from Onyx Pharmaceuticals (Richmond, CA), 12CA5 mAb from Berkeley Antibody Co. (Berkeley, CA). The septapeptide EEYMPME corresponding to the sequence of the EE epitope was synthesized at the Biomolecular Resource Facility at University of California at San Francisco. EE mAb was coupled to protein G-Sepharose as described (23) . EE mAb was biotinylated using the Fluoreporter Biotin-XX labeling kit from Molecular Probes Inc. as described in the manufacturer's instructions. For Western blotting the following dilutions of primary antibodies were used: EE mAb, 3 µg/ml; biotinylated-EE mAb, 1 µg/ml; 12CA5 mAb, 1 µg/ml; anti-G antiserum B600, 1/5,000 dilution; anti-antiserum WO82, 1/250 dilution; anti-antiserum 199.233, 1.2 µg/ml. Secondary antibodies with conjugated horseradish peroxidase were used at a 1/10,000 dilution. Incubations with primary antibodies were from 1 h to overnight, secondary antibodies for 30 min. ECL (Amersham) was used to visualize immunoreactive bands.


RESULTS

Rationale and Initial Studies

One of our major goals was to identify a common site in G protein subunits for introduction of well characterized monoclonal antibody epitopes, in order to improve immunodetection and immunoprecipitation. We scanned sequences of subunits for regions of similar sequence, suitable for introduction of epitopes and predictably accessible to antibodies; we then introduced defined epitopes into these regions and assessed the effects of epitope tags on subunit function. We used the HA (24) and EE (25) epitopes (Fig. 1); these comprise small contiguous stretches of amino acids, are well characterized and have been used for epitope tagging of a variety of proteins (26, 27) .

We introduced epitopes into representatives (, , and ) of three subunit families. Signaling activities of subunit constructs were measured following transient transfection in cell types with a null background, that is, cells in which signaling activity of the corresponding endogenous subunit could be blocked or was absent. For constructs, S49 cyccells provide a genetically null background and signaling activity can be assayed by measuring cAMP production following stimulation of the endogenous -adrenoreceptor (-AR). HEK293 cells provide null backgrounds for assaying coupling of transiently expressed or to the cotransfected -adrenoreceptor (-AR), because the corresponding endogenous subunits do not mediate responses to the -AR in these cells (22, 28) . , like , mediates receptor inhibition of adenylyl cyclase. Because is resistant to PTX, treatment with PTX creates a null background for , by uncoupling endogenous Gfrom activation by receptors (17) . Activities of mutants were therefore assayed by measuring D-dopamine receptor (DR) mediated inhibition of the cAMP accumulation generated by stimulation of the luteinizing hormone receptor (LHR) in CHO K1 cells cotransfected with , LHR, and DR.

Epitope Tagging at Two Internal Sites

Previous work from this laboratory showed that insertion of the HA epitope into a site encoded by an alternative exon (exon 3) of the gene did not disrupt coupling to the -AR or to adenylyl cyclase (18) . Epitope tags at the corresponding positions of and , however, produced non-functional proteins. In addition, appending epitopes at the carboxyl termini of and impaired function (data not shown).

Scanning subunit sequences revealed two regions similar in amino acid composition to both epitopes. The first (site I) is located approximately 125 residues from the amino terminus. Site I sequences are well conserved among members of each subunit family, although overall similarity among subunits at this site is not high (Fig. 1 A). This site appeared suitable for both epitopes. The second region (site II) is located seven residues upstream of the arginine residue in that is ADP-ribosylated by cholera toxin (Fig. 1 B). Site II sequences are highly conserved among all subunits and closely match the EE epitope sequence. In the crystal structure of (29) , site I and site II are located in the helical domain, in loops between helices B and C and E and F, respectively.


Figure 1: Sequences of epitope-tagged subunits. The amino acid sequences of wild-type and epitope-tagged , , , and at site I ( panel A) and site II ( panel B) are shown. Numbers in parentheses refer to the amino acid residues of the wild type sequence that were mutated to create the epitope sequence. The arrow in panel B points to the arginine residue in that is covalently modified by cholera toxin. Panel C lists the amino-terminal sequences of and mutant constructs. Epitope sequences are underlined.



We introduced both epitopes into site I in and measured the ability of epitope-tagged to couple the -AR to stimulation of PI-PLC in HEK293 cells. While the magnitude of stimulation of PI-PLC varied from transfection to transfection, both HA-I and EE-I stimulated PI-PLC to the same extent as did untagged (Fig. 2 A). Agonist dose-response curves for HA-I were similar to that for untagged (Fig. 3 A). Reproducibly, the dose-response curve for EE-I was shifted to the left (Fig. 3 B). Both epitope-tagged proteins were detectable by Western blotting (data not shown).


Figure 3: Agonist dose-response curves for epitope-tagged subunits. Panels A-C, HEK 293 cells were transfected with 1 µg of -AR DNA and 3 µg of pcDNA1 containing the indicated constructs. Production of inositol phosphates in response to varying concentrations of the R agonist, UK 14304, was measured as described under ``Experimental Procedures.'' Panels D and E, CHO K1 cells were transfected using lipofectant with 1 µg of LHR, 2 µg of DR, and 1 µg of pcDNA1 containing the indicated constructs. Production of cAMP in response to 100 ng/ml human chorionic gonadotropin and various concentrations of the DR agonist, quinpirole, was measured as described under ``Experimental Procedures.'' Quinpirole inhibition of cAMP production for cells transfected with pcDNA1 vector was less than 10% of the human chorionic gonadotropin-stimulated cAMP signal (data not shown). Panel F, S49 cyccells were transfected with 10 µg of pSR296 containing the indicated constructs. Production of cAMP in response to various concentrations of the -AR agonist, isoproterenol, was measured as described under ``Experimental Procedures.'' For all panels, each point represents the mean ± S.D. of triplicate measurements. For each panel, at least two additional experiments yielded similar results.



HA-I mediated DR inhibition of adenylyl cyclase but was not detectable by Western blotting using the 12CA5 monoclonal antibody (data not shown). HA-I probably lacked sufficient determinants for recognition by the 12CA5 antibody, because a second HA construct (denoted HA-I), comprising an additional two amino acids of the original HA epitope, was recognized by the 12CA5 antibody on Western blots (data not shown). HA-I mediated DR inhibition of adenylyl cyclase to the same extent as did untagged (Fig. 2 B) and agonist dose-response curves were similar to those for untagged (Fig. 3 D). In contrast to HA-I, EE-I was expressed but coupled weakly to the DR (data not shown). At higher levels of expression, EE-I constitutively inhibited adenylyl cyclase (as does untagged ), suggesting that creation of the EE epitope at site I may perturb activation, perhaps by impairing interactions with and/or receptors.


Figure 2: Signaling activity of subunits. Untagged and epitope-tagged subunits were transiently transfected with the appropriate receptors as described under ``Experimental Procedures'' and the legend of Fig. 3. Panel A, ; panel B, ; panel C, . Effector activities were measured in the absence and presence of maximally stimulating concentrations of receptor agonist and compared to controls transfected with pcDNAI vector (see ``Experimental Procedures''). The extent of effector activation for and constructs is normalized to effector activity in the absence of receptor agonist. For constructs, results are expressed as percent inhibition of the cAMP signal produced by LH receptor stimulation. Values represent the mean ± S.D. of three separate experiments.



Introducing the HA epitope into site I of completely disrupted its signaling activity in either S49 cyccells or in HEK293 cells, even though its expression in HEK293 cells was readily detected on Western blots. Furthermore, cholera toxin failed to activate adenylyl cyclase in S49 cyccells transfected with HA-I (data not shown).

In contrast to epitopes at site I, the EE epitope at site II consistently left signaling activity intact. In response to receptor stimulation, EE-II, EE-II, and EE-II regulated activities of their respective effectors to the same extent as did their untagged counterparts (Fig. 2, A-C). Agonist dose-response curves for EE-tagged constructs were also similar to those of their untagged counterparts (Fig. 3, C, E, and F). All three EE-tagged constructs were easily detected by Western blotting (Figs. 4 and 5). Site II appears to be a potential site for tagging all subunits with the EE epitope, probably because highly conserved sequences at this site closely resemble the sequence of the EE epitope.

Epitope Tags at Site II do Not Alter Membrane Association

We fractionated cells transiently expressing subunits into crude particulate and soluble fractions and measured relative amounts of subunit by immunoblotting. Transiently transfected EE-II, EE-II, and HA-I fractionate like their transiently expressed untagged counterparts and express to the same degree ( Fig. 4and data not shown). Transiently expressed EE-II and untagged are found almost exclusively in the particulate fraction, as is the endogenous . A substantial fraction of transiently expressed EE-II and untagged is soluble, while endogenous is primarily particulate. We suspect that overexpression per se alters the distribution of , because transient transfection of larger amounts of cDNA further increases the fraction of soluble protein (data not shown). The predominantly particulate localization of EE-II (Fig. 7) is similar to that reported by others for untagged (4) . Thus these epitope tags do not appear to alter expression or subcellular distribution of these three subunits.


Figure 4: Subcellular distribution of untagged and epitope-tagged and . CHO K1 cells (5 10) were transfected with 10 µg of untagged , untagged , EE-II, EE-II, or pcDNA1 vector and fractionated into crude particulate and soluble fractions 48 h later, as described under ``Experimental Procedures.'' Equivalent amounts of the particulate ( P) and soluble ( S) fractions were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to a native epitope (WO82) or epitope (199.233). Immunoreactive bands detected in the ``vector'' lanes represent endogenous levels of and in CHO K1 cells.




Figure 7: Subcellular distribution of palmitoylation and myristoylation mutants. CHO K1 cells (5 10) were transfected with 10 µg of pcDNA1 containing the indicated constructs. Crude particulate ( P) and soluble ( S) fractions were prepared following hypotonic lysis 48 h later as described under ``Experimental Procedures.'' Equal volumes of both fractions were analyzed by SDS-PAGE and Western blotting with the EE mAb.



Epitope-tagged Subunits Are Efficiently Immunoprecipitated

Previous work demonstrated that HA-tagged and were efficiently immunoprecipitated by the HA monoclonal antibody (12) . All EE-II tagged subunits immunoprecipitated in a nearly quantitative fashion. A very small fraction of EE-II remained in the supernatant following immunoprecipitation (Fig. 5 A). Efficient immunoprecipitation required the presence of sodium cholate and SDS in addition to Triton X-100. These harsher detergents may be required to effect complete denaturation of the EE epitope when it occurs at sites other than the amino and carboxyl termini of the protein. Immunoprecipitation was specific: no EE-tagged protein immunoprecipitated when a nonspecific antibody was used (Fig. 5 B). Additionally, inclusion in the immunoprecipitation solution of a 7-residue peptide comprising the EE epitope completely blocked immunoprecipitation of EE-II (Fig. 5 A). subunits did not co-immunoprecipitate with subunits tagged with either epitope (Fig. 5 C and data not shown).


Figure 5: Immunoprecipitation of epitope-tagged . CHO K1 cells (5 10) were transfected with 10 µg of EE-II DNA. Crude particulate fractions were prepared following hypotonic lysis 48 h later, as described under ``Experimental Procedures.'' An aliquot was solubilized in 1% cholate and following removal of insoluble material by centrifugation was adjusted to 1% Triton X-100 and 0.5% SDS. In panel A, the soluble extract was divided into 3 equal aliquots. 2 aliquots were immunoprecipitated with EE mAb without or with the presence of a septapeptide (50 µg/ml) corresponding to the EE epitope sequence. Immunoprecipitated material and the residual soluble extract were resolved by SDS-PAGE and probed with biotinylated-EE mAb by Western blotting. Lane 1, starting extract; lane 2, immunoprecipitate without septapeptide; lane 3, residual extract without septapeptide; lane 4, immunoprecipitate with septapeptide; lane 5, residual extract with septapeptide. Panels B and C, the soluble extract was divided into 3 equal aliquots. 2 aliquots were immunoprecipitated with EE mAb or 12CA5 mAb. Equivalent amounts of the immunoprecipitates were resolved by SDS-PAGE and probed with biotinylated EE mAb ( panel B) or G antiserum ( panel C) following Western blotting. Lane 1, starting extract; lane 2, immunoprecipitate with EE mAb; lane 3, immunoprecipitate with 12CA5 mAb.



Myristoylation and Palmitoylation of Epitope-tagged EE-II

We constructed a series of mutants of EE-II (hereafter denoted ) in which we replaced with alanine residues the Glyand Cysresidues that are sites for myristoylation and palmitoylation (Fig. 1 C). We examined the effects of these mutations on the ability of to be myristoylated and palmitoylated, to attach to cell membranes, and to transmit signals from receptors to effectors.

Mutant constructs, transiently expressed in CHO K1 cells, were metabolically labeled with [H]palmitate or myristate. Incorporation of radiolabel into subunits was detected following immunoprecipitation, SDS-PAGE, and fluorography. Initial experiments demonstrated that CHO K1 cells rather indiscriminately incorporated radiolabel from both lipids into sites of myristoylation and palmitoylation. The two sites can be distinguished by their sensitivities to treatment with neutral hydroxylamine; the amide bond linking myristate to glycine is resistant while the thioester bond linking palmitate to cysteine is cleaved. All metabolic labeling experiments, therefore, included treatments with hydroxylamine. As controls for the specificity and completeness of the hydroxylamine treatment, we included transfections with EE-II, which contains only a site for myristoylation, and EE-II, which contains only sites for palmitoylation. As anticipated, radiolabel in EE-II derived from either myristate or palmitate was resistant to hydroxylamine, indicating that it was incorporated at the site of myristoylation (Fig. 6, lane 1). Radiolabel incorporated into EE-II was, on the other hand, completely sensitive to hydroxylamine, indicating its incorporation via a thioester linkage (Fig. 6, lane 7).


Figure 6: Palmitate and myristate labeling of mutant constructs. CHO K1 cells (5 10) were transfected with 10 µg of the pcDNA1 constructs EE-II ( lane 1), EE-II ( lane 2), EE-II G2A ( lane 3), EE-II C3A ( lane 4), EE-II G2AC3A ( lane 5), EE-N ( lane 6), or EE-II ( lane 7). 48 h later cells were labeled for 2 h with 0.75 mCi/ml [H]palmitate or [H]myristate, as described under ``Experimental Procedures.'' Following detergent solubilization, extracts were immunoprecipitated with EE antibody and resuspended in SDS sample buffer. Proteins (one-third of each sample) were resolved by SDS-PAGE. Following fixation, identical gels from each labeling condition were soaked for 12 h in 1 M TrisHCl, pH 7.0 ( panels A and C), or 1 M hydroxylamine, pH 7.0 ( panels B and D). Gels were then processed for fluorography. The remaining one-third of each immunoprecipitate was analyzed by SDS-PAGE and Western blotting with biotinylated-EE mAb. A Western blot from the immunoprecipitate from [H]palmitate labeled cells is shown in panel E. Western blots from myristate-labeled cells were similar (data not shown). Autoradiograms exposed for 23 days.



incorporated radiolabel derived from both myristate and palmitate (Fig. 6, lane 2). A sizeable fraction of the radiolabel from palmitate resisted hydroxylamine treatment, as did almost all of the radiolabel from myristate, indicating that label was incorporated into both sites. Introducing the G2A mutation resulted in incorporation of little or no radiolabel from myristate (Fig. 6, lane 3). Reproducibily, however, G2A still incorporated radiolabel from palmitate (Fig. 6, lane 3) although the extent of labeling was substantially less than that seen with ; all of the incorporated radiolabel was completely sensitive to hydroxylamine, indicating that it was attached through a thioester linkage. C3A incorporated radiolabel from both myristate and palmitate, at levels similar to (Fig. 6, lane 4). In contrast to G2A, however, all radiolabel remained attached to C3A following treatment with hydroxylamine, indicating that it was not attached by a thioester linkage. Finally, as expected, mutations that simultaneously abolished the sites for both myristoylation and palmitoylation (G2AC3A, EE-N) completely abolished incorporation of radiolabel from either lipid (Fig. 6, lanes 5 and 6). The simplest interpretation of the data is that Glyand Cysare, respectively, the sites for myristoylation and palmitoylation. Myristoylation does not require the presence of the site of palmitoylation. Loss of the site of myristoylation, however, reduces but does not prevent palmitoylation.

Removal of Sites for Palmitoylation and Myristoylation Alters Association with Membranes

We compared the subcellular localizations of epitope-tagged with those of the G2A, C3A, and G2AC3A mutants. Crude 100,000 g particulate and cytosolic fractions were prepared from transiently transfected CHO K1 cells and the relative distribution of constructs assessed by Western blotting (Fig. 7). Wild type is found almost exclusively in the particulate fraction as is a mutationally activated (Q205L). In contrast, the G2A and G2AC3A mutants are predominantly cytosolic, although a small amount remains in the particulate fraction. The C3A mutant also is more soluble, although a larger fraction remains in the particulate fraction than for the G2A and G2AC3A mutants. Particulate fraction and mutants are almost completely solubilized by 1% cholate (>90%, data not shown).

Removal of Sites for Palmitoylation and Myristoylation Alters Signaling

We next examined effects of these mutations on the ability of to mediate DR inhibition of adenylyl cyclase, by comparing responses in cells transfected with increasing amounts of DNA encoding wild type or the G2A, C3A, G2AC3A mutants (Fig. 8). In the absence of DR stimulation, increasing amounts of constitutively inhibit cAMP production generated by LHR activation. Stimulation of the DR further inhibits cAMP production; DR-stimulated inhibition levels off at 2 µg of DNA (Fig. 8 A). Although all three mutants mediate DR inhibition of cAMP production, their activities differ strikingly. G2A, which lacks the site of myristoylation, generates little constitutive inhibition of cAMP production on its own, even though expression levels are similar to (expression data not shown); the DR inhibits cAMP production in the presence of G2A, but the extent of inhibition is substantially less than for wild type (Fig. 8 B). In contrast, C3A, which lacks the site for palmitoylation, constitutively inhibits cAMP production quite strongly, and is consistently more effective than wild type . At low amounts of cDNA, C3A when compared to wild type produces a greater inhibition of adenylyl cyclase by the DR, although maximal inhibitions are similar (Fig. 8 C). G2AC3A, lacking both sites of fatty acylation, produces a modest constitutive inhibition of cAMP production, which is barely augmented by stimulation of the DR receptor (Fig. 8 D).


Figure 8: Signaling activity of palmitoylation and myristoylation mutants. CHO K1 cells were transfected with 1 µg of LHR DNA, 2 µg of DR DNA, and various amounts of pcDNA1 containing the indicated EE-II constructs. 48 h later, cAMP production was measured as described under ``Experimental Procedures'' on cells treated with 20 ng/ml human chorionic gonadotropin alone ( filled circles) or with 20 ng/ml human chorionic gonadotropin and 10 µ M quinpirole ( open squares) ( panels A-D). Each point represents the mean ± S.D. of triplicate measurements. Axes in panels B-D are the same as in panel A. Expression of constructs was equivalent as assessed by Western blotting (data not shown). Similar results were obtained in three additional experiments.



We examined whether the inhibition of adenylyl cyclase by might be mediated by sequestration of otherwise free complexes that (in the absence of excess ) conditionally activate adenylyl cyclase. We coexpressed LHR with , an subunit that does not interact with adenylyl cyclase but can sequester complexes (30) . Expression of elevated slightly (up to 20%) cAMP levels following LHR stimulation (data not shown). We conclude that free complexes are not conditionally activating adenylyl cyclase following LHR stimulation.

The ability of G2AC3A to inhibit constitutively cAMP production suggested that neither the site of myristoylation or palmitoylation was necessary for interaction with the effector, adenylyl cyclase. To confirm this inference, both sites were removed by introducing the EE epitope at the amino terminus of a constitutively active mutant, Q205L, and the ability of this mutant to inhibit cAMP production was compared to that of Q205L with the EE epitope at site II. Both constructs inhibited production of cAMP to the same extent at the highest amounts of DNA used for transfection. The mutant lacking sites of lipid attachment produced less inhibition at lower concentrations of transfected DNA (Fig. 9), indicating that while fatty acylations enhance effector interactions of , they are not essential for inhibition of adenylyl cyclase.


DISCUSSION

Extending an approach previously initiated with , we have identified a site common to G protein subunits that may be used for insertion of epitope tags. Using epitope-tagged mutants, our experiments establish the critical importance of covalently attached fatty acids for receptor-mediated signaling by and extend and modify previous observations defining the structural requirements for palmitoylation. Finally, our results provide an experimental basis for investigating the roles of palmitoylation and myristoylation in regulating movement of subunits among membrane compartments and interactions with other signaling proteins.

Epitope Tagging

Availability of epitope-tagged constructs will facilitate investigations of subunit structure and function in intact cells. Our findings underline the importance of assessing functional consequences of the introduction of heterologous epitopes. Although individual subunits could be tagged at site I without disrupting function, only site II consistently tolerated epitope tagging without altering expression, membrane localization, or signaling activity. Although site II probably will prove to be a generally useful site for tagging other subunits with the EE epitope, it will be important to assess function of each new epitope-tagged construct.

The crystal structure of GTP assigns the locations of site I and site II to loops between helices in the helical domain (29) . While this domain contributes importantly to GTP binding and GTP hydrolysis in subunits (31) , the altered functions of subunits tagged at site I hint at additional functions for the helical domain. The shifted dose-response curve produced by EE-I for -AR activation of PLC- and the substantial impairment of DR mediated inhibition of adenylyl cyclase by EE-I suggest that altering site I perturbs interactions, GTP-induced conformational change, or effector activation. Although the crystal structures of (29, 32) do not reveal GTP-induced conformational changes at site I, a recent report of the crystal structure of notes in passing that this region may undergo conformational change (33) . The data presented here argue strongly that the conformation of the region encompassing site I in , , and is functionally important.

Palmitoylation and Myristoylation of

Our results confirm previous observations that is palmitoylated and myristoylated (2, 4, 8) and agree with conclusions based on experiments with other subunits that myristoylation is not affected by mutations that alter the site of palmitoylation (14, 34) . In contrast to the results of others (4, 14) , however, we find that palmitoylation can take place in the absence of the site of myristoylation, although the G2A mutation does reduce the amount of palmitate label detected.

The G2A mutation could alter palmitoylation in several ways. First, incorporation of radiolabel into G2A would be decreased if Glyor its attached myristate were an important structural determinant for palmitoylation. This seems unlikely, however, because both and are palmitoylated even though neither is myristoylated, and lacks an amino-terminal Gly residue.

Second, the G2A mutation could reduce incorporation of palmitate into by impairing its localization to the compartment, presumably a membrane compartment, that contains the relevant palmitoyl transferase. In this view, myristoylation could play an important role in palmitoylation by increasing associations of with cellular membranes directly or by increasing affinity of for complexes, which in turn present to the membrane. The presence of myristate on does increase its association with membranes (Fig. 7) and myristate is reported to increase the affinities of both (5) and (6) for .

Lastly, the lower amount of radiolabel in G2A may reflect more rapid depalmitoylation. Physiologically, Glyand/or myristate may serve an important regulatory role in depalmitoylation, although our data do not address this directly. Glyand/or myristate could also play an important but nonphysiological role in stabilizing palmitate during the extraction and immunoprecipitation procedure. Cell extracts contain palmitoyl thioesterase activity; a substantial loss of palmitate from can take place after breaking cells, during the process of extraction and immunoprecipitation (15) . If the G2A mutation accelerates the rate of depalmitoylation, substantially less palmitate will be detected by the end of the processing procedure, even though the extent of palmitoylation in the intact cell may have been equal in wild type and mutant . This kind of nonphysiological depalmitoylation could also explain the discrepancy between our results and reports that G2A mutants are not palmitoylated. The conditions used here represent a minimal time for extraction and complete immunoprecipitation (2 h). Others, using transiently overexpressed , , or in HEK293 and COS cells (4, 14) , employed lengthier immunoprecipitation protocols and did not detect palmitoylation in G2A mutants. Our ability to detect palmitoylation of G2A in CHO K1 cells may reflect physiologically important cell-specific differences, but may also indicate that extraction and immunoprecipitation protocols are not always reliable for detecting the presence of palmitate.

Membrane Attachment and Signaling Activity

The simultaneous abolition of myristoylation and palmitoylation in produces effects similar to abolition of palmitoylation in (12) . The subcellular localization of both subunit constructs shifts to the soluble fraction; only a small amount remains in the particulate fraction. Receptor mediated interactions with adenylyl cyclase are severely impaired or abolished. Constitutively activating forms of both subunits, however, still productively interact with adenylyl cyclase, albeit less efficiently. The more pronounced functional impairment seen with receptor activation may reflect the critical importance of fatty acid acylation for interactions with complexes which in turn are required for receptor-catalyzed GDP/GTP exchange.

Blocking myristoylation reduced the fraction of associated with the particulate fraction and impaired but did not prevent signal transmission, which requires productive interactions of with , receptor, and effector. These results were surprising because myristoylation of some family members greatly increases their affinity for (5, 6) and is required for productive interaction of with adenylyl cyclase (7) ; this latter finding is in agreement with results of our own experiments with a constitutively active form of in intact cells.() Activation of non-myristoylated , whether by receptor (G2A) or by a constitutively activating mutation (EE-N Q205L), still resulted in inhibition of adenylyl cyclase. Larger amounts of the nonacylated, mutationally activated were required for maximal inhibition of adenylyl cyclase (Fig. 9). Presumably, myristate increases either the affinity of for adenylyl cyclase or the amount of at the plasma membrane, thereby increasing its effective concentration.


Figure 9: Signaling activity of Q205L palmitoylation and myristoylation mutant. CHO K1 cells were transfected with 1 µg of LHR DNA and various amounts of pcDNA1 containing EE-II ( open squares), EE-II Q205L ( filled squares), or EE-N Q205L ( filled circles) constructs. 48 h later, cAMP production was measured as described under ``Experimental Procedures'' on cells treated with 20 ng/ml human chorionic gonadotropin. Each point represents the mean ± S.D. of triplicate measurements. Expression of constructs was equivalent as assessed by Western blotting (data not shown). An additional experiment yielded similar results.



Mutation that prevents palmitoylation decreases substantially the fraction of associated with the particulate fraction but surprisingly this has no discernible effect on DR-mediated inhibition of adenylyl cyclase (compare DR inhibition of cAMP production for versus C3A, Fig. 8, A versus C). Even more surprisingly, preventing palmitoylation of enhances its constitutive inhibition of adenylyl cyclase. This effect is seen most clearly in comparing myristoylated forms of (compare LHR stimulation of cAMP production in the presence of versus C3A, Fig. 8, A versus C), but also occurs in the absence of myristoylation (compare LHR stimulation of cAMP production in the presence of G2A versus G2AC3A, Fig. 8, B versus D).

The mechanism underlying this phenomenon is obscure. The constitutive activity of could reflect the presence of GTP that arises from non-receptor catalyzed exchange of GDP for GTP. In this context, palmitoylation could decrease the rate of GDP/GTP exchange directly by generating a more stable conformation of GDP or indirectly by increasing the affinity of for . Although myristoylation can indirectly slow exchange by increasing the affinity of for , no evidence yet implicates palmitoylation or any other fatty acylation of subunits in modulating GDP/GTP exchange.

Alternatively, palmitoylation could decrease constitutive activity by directing to a membrane domain that regulates interactions with effector. Increasing numbers of signaling molecules, including G protein subunits, are being found to concentrate in caveolae (35, 36) , plasmalemmal domains that play roles in intra- and intercellular communication (37) . Palmitoylation is a signal that directs otherwise cytosolic proteins to caveolae, as shown most clearly for kinases of the Src family (38) . It is attractive to hypothesize that palmitoylation of localizes it to caveolae and that this localization prevents spurious interactions with effector.

Based on experiments with (12, 15, 18) , Wedegaertner and Bourne (15) proposed that the dynamic palmitoylation cycle regulates signaling by Gand, possibly, by other G proteins. Depalmitoylation of follows its activation by receptor and correlates with its movement from the membrane into the soluble compartment. After hydrolysis of GTP, GDP is repalmitoylated, binds , and returns to the membrane for another cycle of receptor activation (although the temporal sequence of events that follows GTP hydrolysis is unknown). Depalmitoylated is functionally uncoupled from receptor activation, so that depalmitoylation may be viewed as a potential mechanism for decreasing the signaling activity of .

While it is reasonable to postulate a palmitoylation cycle for , depalmitoylation does not seem likely to decrease -dependent signaling, at least in relation to adenylyl cyclase. First, the subcellular distribution of mutationally activated is virtually identical to that of non-activated ; in contrast, constitutive activation shifts a large fraction of into the soluble compartment (12) . Second, receptor mediated signaling by is enhanced by mutation that prevents palmitoylation, again in contrast to the effect this mutation has on signaling. Third, mutational removal of the palmitoylation site in produces a protein whose ability to constitutively inhibit adenylyl cyclase is actually increased. Dynamic palmitoylation of may serve to shuttle it between membrane compartments, rather than to decrease its activity.

Summary

Our results sketch a low resolution picture of the roles that palmitate and myristate play in the membrane association and signaling properties of . The lack of resolution reflects the fact that our assays are all-inclusive measurements of signaling function, and do not distinguish individual steps during receptor-mediated signal transduction, such as binding to and binding to receptor. Nevertheless our results clarify the distinct and different effects of palmitoylation and myristoylation on different G protein subunits, and raise new questions which can be tackled with epitope-tagged constructs.


FOOTNOTES

*
This work was supported by a National Alliance for Research on Schizophrenia and Depression Young Investigator Award (to P. T. W.), National Institute of Mental Health Grant MH00961 (to P. T. W.), National Institutes of Health Grants GM-27800 and CA-54427 (to H. R. B.), and a grant from the March of Dimes (to H. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: S-1212, Box 0450, UC Medical Center, San Francisco, CA 94143. Tel.: 415-476-8161; Fax: 415-476-5292.

The abbreviations used are: PTX, pertussis toxin; HA, hemagglutinin; EE, Glu-Glu; -AR, -adrenergic receptor; LHR, luteinizing hormone receptor; DR, D-dopamine receptor; -AR, -adrenergic receptor; PI-PLC, phosphoinositide-specific phospholipase C; MEM, minimal essential medium; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

P. T. Wilson and H. R. Bourne, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Eva Schindler for dedicated technical assistance and Susan Munemitsu, John Lyons, Bonnee Rubinfeld, and Phil Wedegaertner for useful advice. We thank Pablo Garcia for the EE cDNA construct and Gordon Chew for HA cDNA constructs.


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