PAR1 Thrombin Receptor-G Protein Interactions

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 4, Issue of January 28, pp. 2627–2635, 2000 Printed in U.S.A.

PAR1 Thrombin Receptor-G Protein Interactions SEPARATION OF BINDING AND COUPLING DETERMINANTS IN THE G␣ SUBUNIT* (Received for publication, July 28, 1999, and in revised form, September 16, 1999)

Steven Swift, Paul J. Sheridan, Lidija Covic, and Athan Kuliopulos‡ From the Molecular Cardiology Research Institute, Division of Hematology/Oncology, New England Medical Center and Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Thrombin plays a central role in blood coagulation and in the cellular response to injury, inflammation, and wound repair. Many of these complex cellular events are initiated by thrombin cleavage of the protease-activated receptor-1 (PAR1)1 at residues Arg41-Ser42 located within the N-terminal extracellular domain. The new N terminus, Ser42-Phe-Leu-Leu-ArgAsn47, serves as an intramolecular ligand that binds and activates the body of the PAR1 thrombin receptor (1–3). Peptides corresponding to the freshly cleaved N terminus can also activate PAR1 by an intermolecular mechanism (1). A number of different guanine nucleotide-binding (G) protein ␣-subunits have been shown to couple to PAR1, including members of the Gi, Gq/16, and G12/13 families, but the relative importance of these interactions is not well understood (4 – 8). Recent PAR gene knockout experiments in mice and cloning of two addi* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Supported by National Institutes of Health Grants R29GM52926 and R01HL57905, and by Scholar Awards from the Pew Scholars Program in the Biomedical Sciences and the American Society of Hematology. To whom correspondence should be addressed. Tel.: 617-6368482; Fax: 617-636-4833; E-mail: [email protected]. 1 The abbreviations used are: PAR1, protease-activated receptor-1; bp, base pair(s); PCR, polymerase chain reaction; IDA, iminodiacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTP␥35S, guanosine 5⬘-O-␥-(35thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase; TRAP, thrombin receptor agonist peptide. This paper is available on line at http://www.jbc.org

tional thrombin receptors (PAR3 and PAR4) and trypsin/ tryptase receptor (PAR2) have shown that different tissues possess a distinct complement of protease receptors that together determine their respective responses to a variety of proteolytic inputs (9 –13). A divergence point in protease receptor signaling is that activation of any particular G␣䡠␤␥ complex will produce two effectors, namely activated G␣ and free ␤␥ (14). For instance, since phospholipase C-␤ can be potentially activated by either Gq or ␤␥, it is conceivable that non-Gq subunits may serve the negative function of sequestering ␤␥ to prevent constitutive activation of the phospholipase C-␤ pathway (15). In platelets, it now seems likely that Gq is the primary effector for phospholipase C-␤ rather than free ␤␥. This was demonstrated in Gq gene knockout mice (7), whose platelets lack thrombin-dependent phosphoinositide responses or the ability to aggregate. The Gq (⫺/⫺) platelets retain partial function since they can undergo thrombin-dependent shape change. Therefore, other G␣ subunits such as Gi2 and G12/13, must necessarily serve as primary effectors for the dramatic cytoskeletal rearrangements that are a prerequisite for platelet aggregation (16). Other functions of platelet thrombin receptors are influenced by Gi-dependent depression of cAMP levels. The cAMP levels in platelets modulate the activity of platelets; high cAMP levels dampen the response of platelets to agonists and cause attenuation of intracellular Ca2⫹ levels (17) in part by stimulating Ca2⫹ efflux by activation of the plasma membrane Ca2⫹ATPase (18). The role of Gi in PAR1 responses has been shown by specific pertussis toxin blockade of Gi which significantly reduces SFLLRN-stimulated GTPase activity (19). It is not known how the Gi family members individually regulate adenylyl cyclase, however, the relative abundance is Gi2 ⬎⬎ Gi3 ⬎ Gi1 in platelets (20). One of the major unanswered questions of thrombin receptor signaling is why does PAR1 couple to only a subset of the available G protein partners in a given cell? Presently, there is limited information regarding the molecular determinants of PAR1-G protein interactions. A single study by Coughlin and colleagues (21) involved replacing internal cytoplasmic loops of the Gs-coupled ␤2-adrenergic receptor and the Gi-coupled dopamine D2 receptor with the analogous loops from PAR1. They found that chimeric ␤2-adrenergic and dopamine D2 receptors bearing the second intracellular loop (i2) from the PAR1 acquire Gq-coupling capability. This led them to conclude that the PAR1 i2 loop may provide “coupling instructions” for Gq specificity in several different receptor contexts but specific PAR1-Gi subunit interactions have yet to be elucidated. With limited high resolution structural data available for receptor-G protein interfaces (22), most of the focus on the general molecular determinants of coupling preferences centers on mutagenesis of receptor i3 loops and the extreme C-terminal residues of G␣ subunits (23). For instance, point mutations in the i3 loop of

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Signal transfer between the protease-activated PAR1 thrombin receptor and membrane-associated heterotrimeric G proteins is mediated by protein-protein interactions. We constructed a yeast signaling system that resolves domain-specific functions of binding from coupling in the G␣ subunit. The endogenous yeast G␣ subunit, Gpa1, does not bind to PAR1 and served as a null structural template. N- and C-terminal portions of mammalian Gi2 and G16 were substituted back into the Gpa1 template and gain-of-function assessed. The C-terminal third of G16, but not of Gi2, provides sufficient interactions for coupling to occur with PAR1. The N-terminal two-thirds of Gi2 also contains sufficient determinants to bind and couple to PAR1 and overcome the otherwise negative or missing interactions supplied by the C-terminal third of Gpa1. Replacement of the N-terminal ␣-helix of Gi2, residues 1–34, with those of Gpa1 abolishes coupling but not binding to PAR1 or to ␤␥ subunits. These data support a model that the N-terminal ␣N helix of the G␣ subunit is physically interposed between PAR1 and the G␤ subunit and directly assists in transferring the signal between agonist-activated receptor and G protein.

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PAR1 Thrombin Receptor-G Protein Interactions

EXPERIMENTAL PROCEDURES

Materials—Human ␣-thrombin (3433 NIH units/mg) was from Haematologic Technologies (Essex Junction, VT). Restriction enzymes and Vent DNA polymerase were from New England Biolabs (Beverly, MA). Plasmid Construction—The ste2::URA3-hisG disrupter construct, pSte2KO, was assembled from 5⬘ (1480 bp) and 3⬘ (700 bp) flanking sequences of STE2 with the 5000-bp hisG-URA3-Kanr-hisG fragment from pSE1076 (gift of E. Elion) inserted between them in pUC19. The STE2 expression vector, p112-H6T7Ste2⌬300, is derived from the 2-␮m TRP1 plasmid, YEplac112 (33). The 5⬘-untranslated region of STE2 was amplified by PCR to create a 544-bp SphI-XbaI fragment that was placed into corresponding sites of pUC19 and designated p19 – 5⬘UTRH6. The fragment of the STE2 gene encoding amino acid residues Ala4 to Thr297 was amplified by PCR and placed into the XbaIKpnI sites of p19 –5⬘UTRH6 to create p19-H6T7Ste2. PCR was then used to amplify 200 bp of the 3⬘-untranslated region of STE2. This 200-bp fragment was ligated into KpnI-EcoRI sites of p19-H6T7Ste2 to create p19-H6T7Ste2⌬300. The 1680-bp SphI-EcoRI fragment was then shuttled into the corresponding sites of YEplac112 and YEplac195 (2 ␮m URA3 (33)) to create p112-H6T7Ste2⌬300 and p195H6T7Ste2⌬300, respectively. The p112-STE2 vector contains the 2010-bp SphI-EcoRI wild-type STE2 fragment amplified by PCR. The thrombin receptor expression vector, p112-H6PAR1⌬379 was created by inserting the region of hPAR1 encoding residues Leu38Glu377 into the BamHI and KpnI sites of p112-H6T7Ste2⌬300. PCR was used to amplify the cDNA of hPAR1 subcloned into the SalI sites of pBluescript (gift of S. Coughlin). This BamHI-KpnI fragment was subcloned into the analogous sites of p112-H6T7Ste2⌬300 to make p112-

H6PAR1⌬379. An extra two codons (Met378-His379) were added to the C terminus to accommodate a KpnI restriction site. The hPAR1 gene comprising residues Leu38-Tyr420 was amplified with PCR primers (⫹) 5⬘-TACTCTCGCGATCTAGACGCAGGCCAGAATCAAAAGCA-3⬘ and (⫺) 5⬘-TCACTATGCATGGATCCGTATATGCTGTTATCCAGGTTACT-3⬘. A 1205-bp NruI-NsiI fragment was subcloned into the HincII-PstI sites of p19H6T7Ste2⌬300 (1680-bp H6T7Ste2⌬300 fragment subcloned into the SphI-EcoRI sites of pUC19). The 949-bp PstI-KpnI fragment was then subcloned into the corresponding sites of p112-H6PAR1⌬379 to create p112-H6PAR1. This PAR1 construct has the 5 C-terminal amino acids Lys421-Lys-Leu-LeuThr425, replaced with GSMQ. The high copy number yeast expression vector pRS426-GPA1 (URA3 2 ␮m) is the 1900-bp EcoRI fragment of GPA1 with flanking 5⬘ and 3⬘ GPA1 regulatory elements subcloned into pRS426 (gift of S. DeSimone and J. Kurjan). The pPGK-Gi2 vector expresses full-length rat Gi2 and the pPGK-Gi2-gpa1 vector expresses a chimera comprising residues 1–212 of Gi2 joined to residues 330 – 472 of Gpa1 (34). pPGK-␣N-i2 expresses a chimera that fuses Gpa1 residues 1– 42 to residues 35–355 of Gi2. This was made by PCR amplification of the respective regions from the Gpa1 and Gi2 vectors, and subcloning them into pPGK. p195Gpa1-i2 expresses a chimera encoding residues 1–329 of Gpa1 joined to residues 213–355 of Gi2 under the control of the GPA1 promoter in the 2-␮m URA3 plasmid, YEplac195 (33). This was made by PCR amplification of the corresponding GPA1 coding sequence plus 200 bases upstream, ligation to the corresponding Gi2 sequence, and subcloning into YEplac195. p195-Gpa1–16 expresses a chimera encoding residues 1–329 of Gpa1 joined to residues 220 –374 of G16, under the control of the GPA1 promoter. p195-Gpa1–16 was made by replacement of the Gi2 sequence in p195-Gpa1-i2 with the sequence encoding residues 220 – 374 of G16. G16 was amplified from the pCISG16 template by PCR (gift of M. Simon). Construction of Yeast Strains—The yeast strains (Table I) were derived from either W303a or a protease-deficient strain, BJ2168. The chromosomal STE2 allele was deleted by homologous recombination (35) at 5⬘- and 3⬘-flanking regions with pSte2KO. The BamHI-PvuII fragment of pSte2KO containing ste2⌬hisG-URA3-hisG, was used to transform EY957 and BJ2168, and ura3⫺ derivatives were recovered by counterselection on 5-fluoroorotic acid media (36) to yield KY1 and KY15, respectively. The chromosomal G␣ gene, GPA1, was deleted in strain EY1262 (gift of E. Elion) using the knockout construct pSKIPCR-URA3 (gift of S. DeSimone) digested with EcoRI. A ura3 derivative was made by counterselection on 5-fluoroorotic acid media by previously published methods (37) to make KY187 in far1-deficient genetic backgrounds. ␤␥ Sequestration Determinations in Yeast—Yeast strain KY187 (gpa1 ura3 his3 FUS1-HIS3) carrying a 2-␮m URA3 vector with a G␣ subunit gene was grown in synthetic dextrose media deficient for uracil at 250 rpm and 30 °C to ⬃1 ⫻ 106 cells/ml at A600 ⫽ 0.4. Cells were pelleted, washed in sterile distilled water, and resuspended to a concentration ⬃2 ⫻ 104 cells/ml in synthetic dextrose media deficient for uracil and histidine. The cells were grown as above, and assayed for absorbance at 600 nm for 2–3 days. Slow growing cultures were split 2-fold, and histidine was added to a final concentration of 20 mg/liter to one set of cultures. Growth and absorbance assays were continued to stationary phase for the cultures with histidine. Expression of PAR1 and G Proteins in Yeast—Cultures (5 ml) of yeast strains harboring His6T7-tagged PAR1 expressed from high copy number (2 ␮m TRP1) vectors were grown to saturation in CAA-TRP media (1 liter contains 6 g of casamino acids, 2% glucose, 1.7 g of yeast nitrogen base, 5 g of ammonium sulfate, 40 mg of adenine, and 20 mg of uracil) at 30 °C. If coexpression of PAR1 and G-protein was required, the G-protein was expressed from a second vector (2-␮m URA3) using media lacking uracil and tryptophan. A dilution of 1:1000 of the overnight culture was made into fresh CAA-TRP (or CAA-TRP-URA) media and grown at 30 °C to a density of 1–1.5 A600. All further steps were carried out at 4 °C. Cells from 2-liter cultures were harvested by centrifugation for 30 min at 3,300 ⫻ g, and resuspended in 200 ml of lysis buffer (20 mM KPO4, pH 7.0, 1 mM MgCl2, 10% glycerol, 1 mM EDTA, and 1⫻ protease inhibitor mixture (38)). Glass beads (0.5 mm) were then added to the cell suspension (50% v/v) and cells were disrupted in a stainless steel bead beater (Biospec, Bartlesville, OK) equipped with ice-water jacket. The cell lysate was centrifuged for 30 min at 8,000 ⫻ g and the supernatant (180 ml) subjected to ultracentrifugation at 150,000 ⫻ g for 1 h. The crude membrane pellets were resuspended in 10 ml of storage buffer (20 mM KPO4, pH 7.5, 100 mM NaCl, 10% glycerol) by sonication for 1 min at a power setting of 4 –7 on a Branson sonicator and stored as 1 -ml aliquots at ⫺80 °C. Yeast membrane


the human muscarinic m2 receptor can compensate for point mutations at C-terminal residues of G␣ (24) consistent with direct intermolecular contact occurring between these regions. Because there are now high resolution x-ray structures of G␣␤␥ heterotrimers (25, 26), it is possible to genetically map the solvent-accessible surface of the G protein for the complicity of specific residues in receptor binding and activation. The first such study conducted on a large scale consisted of 100 alanine-substituted solvent-exposed residues of Gt that were tested biochemically for impaired interactions with rhodopsin (27). Only 9 of the 100 substituted residues were believed to affect solely receptor binding. These sites mapped to the Cterminal 41 amino acids of Gt which contains the ␤6/␣5 motif and crucial 11-residue C-tail which are known to mediate receptor-G␣ interactions (28). A genetic study of the C-terminal 50 residues of the yeast G␣ subunit, Gpa1, correlated well with the mammalian data (29). The potential yeast receptor-Gpa1 interface mapped to 4 of the same 9 residues that were identified in Gt with an additional 11 residues that were located in the ␣5 helix and C-tail. Thus at a genetic level, it appears that the general receptor-G␣ contact surface shares similar architectural features between yeast and mammals. Given the inherent difficulty in studying structure-function relationships of specific PAR1-G protein complexes in mammalian cells, we developed a more simplified receptor-G protein yeast model system (30 –32) that could be used to elucidate mechanistic details of PAR1-G protein coupling. In the studies presented here, we have replaced the endogenous yeast receptor with the human PAR1 thrombin receptor and obtained high affinity agonist binding and functional coupling to co-expressed G proteins. Determinants of PAR1-G protein coupling are separable from binding activity and are found throughout a large interface that encompasses both N- and C-terminal regions of the G␣ subunit. Our data are also consistent with a model that the N-terminal ␣N helix of the G␣ subunit inserts between PAR1 and the G␤ subunit and mediates binding and coupling between agonist-activated receptor and G protein. These genetic and biochemical studies further underscore the advantage of using yeast where one can reconstitute a simple signaling unit consisting of ligand-receptor-G-protein that can be tested for functional properties in isolation from all other mammalian proteins.


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TABLE I Yeast strains Strain

Genotype

Reference ⫹

MATa STE2 GPA1 ura3–1 leu2–3,112 trp1–1 his3–11,15 ade2–1 Gal MATa STE2 GPA1 leu2 trp1 ura3–52 prb1–1122 pep4–3 prc1–407 gal2

R. Rothstein Ref. 57

Isogenic derivatives of W303a EY957 KY1 KY33 KY69 KY70 EY1262 KY50 KY51 KY64 KY91 KY92 KY103 KY114 KY175 KY181 KY187 KY321

sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬ sst1⌬

Ref. 58 This study This study This study This study Ref. 56 This study This study This study This study This study This study This study This study This study This study This study

Isogenic derivatives of BJ2168 KY15 KY27 KY34

ste2⌬ ste2⌬ ⫹ p112-H6Ste2⌬300 ste2⌬ ⫹ p112-H6T7Ste2⌬300

ste2⌬ ste2⌬ ⫹ p112-H6T7Ste2⌬300 ⫹ pSB234 ste2⌬ ⫹ p112-H6PAR1 ste2⌬ ⫹ p112-H6PAR1⌬379 far1⌬ lys2::FUS1-HIS3 his3⌬200 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ p112-STE2 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ p112-H6PAR1 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ p112-H6PAR1 ⫹ pPGK-Gi2-gpa1 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ p112-H6PAR1 ⫹ pPGK-Gi2 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ p112-H6PAR1 ⫹ pRS426-GPA1 far1⌬ lys2::FUS1-HIS3 his3⌬200 ste2⌬ ⫹ pPGK-Gi2 far1⌬ lys2::FUS1-HIS3 his3⌬200 gpa1⌬ ⫹ p112-H6PAR1 ⫹ p195-Gpa1-i2 far1⌬ lys2::FUS1-HIS3 his3⌬200 gpa1⌬ ⫹ p112-H6PAR1 ⫹ p195-Gpa1–16 far1⌬ lys2::FUS1-HIS3 his3⌬200 gpa1⌬ far1⌬ lys2::FUS1-HIS3 his3⌬200 gpa1⌬ ⫹ p112-H6PAR1 ⫹ pPGK-␣N-i2

proteins were maintained at 10 –15 mg/ml for stability. Ni-chelation Chromatography of His6-tagged PAR1-G Protein Complexes—A 5-ml iminodiacetic acid (IDA)-Sepharose column (Novagen) was charged with 10 ml of 100 mM NiSO4 and equilibrated with 50 ml of 5ImBB (5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9). Typically, 12 mg of crude yeast membrane protein containing H6PAR1/ G-protein was applied to the IDA affinity column and recycled for 2 h at 0.38 ml/min. Bound H6PAR1/G-protein was washed with 25 ml of 5ImBB/0.1C (5ImBB ⫹ 0.1% CHAPS, w/v), and eluted with 15 ml of 100ImBB/0.1C (100 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9, 0.1% CHAPS, w/v). Alternatively, 0.1% SDS was substituted for 0.1% CHAPS in the wash (5ImBB/0.1S) and elution buffers (100ImBB/ 0.1S). The affinity purified H6PAR1/G-protein was dialyzed against 2 ⫻ 4 liters of 0.05% SDS, 5 mM Tris-HCl, pH 7.5, in 12–14-kDa Mr cut-off dialysis bags. 1-ml aliquots of dialyzed H6PAR1/G-protein were transferred to 1.6-ml polypropylene tubes, evaporated to dryness with a Speed-Vac concentrator (Savant), and stored at ⫺20 °C. These dried fractions were reconstituted in Laemmli sample buffer and resolved by 10% SDS-PAGE and Western analysis. Radioligand Binding Assays—The high affinity peptide agonist, 56 (Ala-(pF-Phe)-Arg-cyclohexylalanine-homoarginine-Tyr-NH2) was a gift from Merck (39). Compound 56 was iodinated on tyrosine and purified to radionuclide purity by high performance liquid chromatography by previously described methods (40). The 125I-56 peptide was dissolved in 10% dimethyl sulfoxide, 90% 50T/0.1C (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1% CHAPS, w/v) as a 50 nM stock solution just prior to use. TRAP peptide (SFLLRN-NH2), and NAc-Ala-(pF-Phe)-Arg-cyclohexylalanine-Arg-Tyr-NH2 (NAc-TRAP) were synthesized and purified by M. Berne in the Tufts University Analytical Core Facility. Peptides were stored as 50⫻ stock solutions in dimethyl sulfoxide at ⫺20 °C. His6T7-tagged receptor-G-protein complexes were partially purified from crude yeast membranes using a ratio of 200 ␮g of loaded protein/25 ␮l of Ni-IDA-Sepharose beads. Typically, 5 mg of membrane protein at 10 –15 mg/ml was added to 625 ␮l of Ni-IDA beads in 6.25 ml of 5ImBB. The mixture was rocked at 4 °C for 1 h and washed with 2 ⫻ 14 ml of 5ImBB/0.1C, then 2 ⫻ 14 ml of 50T/0.1C. The washed Ni IDA-H6T7receptor(G protein) beads were suspended in 6.25 ml of 50T/0.1C, and 250-␮l aliquots distributed into 1.6-ml polypropylene tubes. Binding assays were done in 500-␮l systems containing 25 ␮l of Ni-IDAH6PAR1(G protein) beads, 1 nM to 1 mM agonist/antagonist peptide, 0.7– 0.9 nM 125I-56, 2.1% dimethyl sulfoxide (v/v) in 50T/0.1C. The binding mixtures were rocked at 4 °C for 30 min and terminated by centrifugation at 12,000 ⫻ g ⫻ 30 s. The supernatants containing unbound 125I-56 were removed and 10 ␮l counted in a Packard 5550 ␥-scintillation counter. The beads were resuspended in 1 ml of ice-cold 50T/0.1C, filtered over Whatmann 25-mm GF/C glass fiber filters (presoaked in 0.3% polyethylenimine-50T/0.1C), washed 2 ⫻ with 5 ml of 50T/0.1C, and filter-bound 125I-56 counted.

This study This study This study GTP␥S Binding Assays—We adapted the assay used for mammalian cells (41) to our yeast system with a few modifications. The H6PAR1(Gprotein) complexes were prepared as above except we used a ratio of 175 ␮g of loaded protein per 15 ␮l of Ni-IDA beads and two washes with 14 ml of TE-HCl (10 mM triethanolamine-HCl, pH 7.5) instead of 50T/0.1C. Assays were performed in 200-␮l volumes and consisted of 15 ␮l of Ni-IDA-H6PAR1(G-Protein) beads suspended in 150 ␮l of TE-HCl, 5 ␮l of 0.25 nM to 250 ␮M peptide agonist in 20% dimethyl sulfoxide, 80% TE-HCl, and 40 ␮l of 5⫻ AB (250 mM TE-HCl, 25 mM MgCl2, 5 mM EDTA, 5 mM dithiothreitol, 500 mM NaCl, 2.5 nM GTP-␥-35S), ⫾ 5 ␮l of 10 ␮M GDP in TE-HCl. Reaction mixtures were incubated at 30 °C for 1 h in 10 ⫻ 75-mm polypropylene tubes with shaking at 250 rpm. Assays were terminated by addition of 2.5 ml of ice-cold WB (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2) and filtered through 25-mm GF/C filters. Filters were washed with 2 ⫻ 2.5 ml of WB and bound GTP-␥-35S quantitated by ␤-scintillation counting as described (41). Antibodies and Flow Cytometry—The polyclonal antibody (SFLLRAb) directed against the PAR1 ligand region, residues S42FLLRNPNDKYEPF55C, was generated as described previously (42). The T7-tag mouse monoclonal antibody (T7-Ab) directed against residues MASMTGGQQM was purchased from Novagen. AS-Ab is the rabbit polyclonal antibody (NEN Life Science Products Inc.) raised against the Gt Cterminal peptide, KENLKDCGLF, which reacts with Gi2 (43). Immunoblotting was done as described previously (44). The Gpa1-Ab is a rabbit polyclonal antibody made against full-length Gpa1 (45). In flow cytometry experiments, yeast cells were fixed in 5% formaldehyde for 2 h at 25 °C prior to fluorescent staining. After 2 successive washes with WB2 (100 mM K2HPO4, 100 mM KH2PO4, 1.2 M sorbitol) ⬃107 cells were resuspended in 1 ml of WB2 containing 88 units of Lyticase (Roche Molecular Biochemicals) and incubated for 30 min at 30 °C in a rollodrum to remove the yeast cell wall and form spheroplasts. Intact spheroplasted cells were stained with either the T7-Ab (final concentration 1 ␮g/ml), or SFLLR-Ab (final concentration 2 ␮g/ml) for 30 min at 25 °C in the presence or absence of 0.1% Tween 20. This was followed by incubation with secondary antibody for 30 min at 25 °C using either goat anti-mouse IgG-fluorescein isothiocyanate (10 ␮g/ml; DAKO) or goat anti-rabbit IgG-fluorescein isothiocyanate (6 ␮g/ml; Zymed Laboratories Inc.). Cells were analyzed for fluorescence with a FACScan flow cytometer (Becton Dickinson). RESULTS AND DISCUSSION

Expression and Thrombin Cleavage of PAR1 in Yeast Membranes—PAR1 is a type I integral membrane protein which has a cleavable N-terminal hydrophobic signal sequence that properly orients the receptor in the lipid bilayer (1). The type III endogenous Ste2 ␣-mating factor receptor uses the first transmembrane ␣-helix as a signal peptide which is not cleaved by a


W303a BJ2168

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signal peptidase (46). To assure correct topology and stable expression, we converted PAR1 from a type I to a type III membrane protein. The hydrophobic signal sequence was replaced with a hydrophilic His6-T7 tag at the N terminus of PAR1 (H6PAR1). PAR1 was expressed in a high copy number vector under the transcriptional control of the STE2 gene. Yeast protein extracts were prepared and probed with a monoclonal anti-T7 antibody (T7-Ab) and a polyclonal antibody (SFLLR-Ab) directed against PAR1 residues Ser42-Phe55. As shown in Fig. 1, lane 5, H6PAR1 localizes to the membrane fraction (UP) and has a relative molecular mass of 34 kDa. The SFLLR-Ab also stains three endogenous yeast proteins migrating at 66, 49, and 46 kDa. Noncovalent higher order H6PAR1 species such as dimer (70 kDa) and tetramer (150 kDa) resist disassociation by 1% SDS as has been observed with other receptors (47) but can be minimized by the inclusion of 4 M urea in the sample buffer (Fig. 1, lane 7). Since the observed molecular mass is 12 kDa smaller than expected (46 kDa) we wanted to rule out C-terminal proteolysis or premature translation termination of the heterologously produced PAR1. To address this mass discrepancy, we expressed a shortened version of the receptor (H6PAR1⌬379) that lacked the C-terminal cytoplasmic domain, residues 380 – 425. As shown in Fig. 1, lane 12, the H6PAR1⌬379 is also targeted to the membrane fraction and has a monomeric molecular mass of 30 kDa which is 4 kDa smaller than the nearly full-length H6PAR1 (expected ⌬molecular mass is 5 kDa). This indicates that PAR1 is full-length but migrates anomalously fast on SDS-polyacrylamide gel electrophoresis. Extensive digestions of H6PAR1 with glycopeptidase F failed to show any shift in size thereby ruling out N-linked glycosylation (data not shown).

One test of PAR functionality is the ability of the cognate protease to recognize and cleave the receptor at the correct cleavage site. As shown in Fig. 1, we demonstrated that yeastexpressed PAR1 can be cleaved at the Arg41-Ser42 peptide bond by nanomolar thrombin. Treatment of H6PAR1 with 5 nM thrombin results in essentially complete loss of the T7-epitope (Fig. 1, lane 8) and the expected slight downward shift in mass is detected by the SFLLR-Ab (Fig. 1, lane 6). Likewise, cleavage of H6PAR1⌬379 at the Arg41 site with 1 nM thrombin causes ⬎97% loss of the T7-epitope (Fig. 1, lane 13). Further characterization of plasma protease cleavage sites and cleavage kinetics of the yeast-produced receptor are presented in a separate report (42). Cellular Localization of PAR1 in Yeast—Flow cytometry was used to determine whether PAR1 is targeted to the yeast plasma membrane and in the proper orientation. As positive and negative controls, we first examined the localization of H6T7Ste2 (KY34) and H6Ste2 (KY27) which lacks the T7 epitope. As shown in Fig. 2C, the median fluorescence intensity of KY34 increased 1.5-fold relative to KY27 in the absence of Tween 20. When these cells were permeabilized with 0.1% Tween 20, the median fluorescence intensity increased to 2.6fold relative to KY27. Therefore, the further increase in fluorescence of the Tween 20 permeabilized KY34 cells relative to the intact KY34 cells indicates that the highly expressed H6T7Ste2 is distributed both on plasma membrane and within membrane compartments located in the cytoplasm. Examination of T7-Ab-stained KY34 by in situ immunofluorescence showed that H6T7Ste2 stains as a uniform coating on the plasma membrane and as bright-stained objects within the cytoplasm (data not shown). The cellular distribution of


FIG. 1. Expression and proteolytic cleavage of PAR1 in yeast membranes. The top is a schematic of the His6T7-tagged PAR1 (H6PAR1) used for heterologous expression in yeast. H6PAR1 terminates at amino acid residue 424; H6PAR1⌬379 lacks cytoplasmic tail amino acid residues 380 – 425. The amino acid residue numbers starting with Leu38 are those of the human PAR1. The bottom is anti-SFLLR and anti-T7 Westerns of yeast extracts separated by 10% SDS-PAGE. Lanes 1, 2, and 9 represent yeast extracts from KY51 (Ste2). Lanes 3– 8 represent yeast extracts from KY69 (H6PAR1). Lanes 10 –13 represent yeast extracts from KY70 (H6PAR1⌬379). The thrombin receptors and yeast mating factor receptor (Ste2) were expressed on a high copy number vector (2 ␮m TRP1) under the transcriptional control of the STE2 gene. UP is the yeast membrane protein fraction (pellet) obtained by ultracentrifugation of total cellular protein; US is the soluble protein fraction (supernatant) obtained by ultracentrifugation of total cellular protein; WC is the whole cellular protein extract obtained by glass bead lysis of yeast. Protease digestions were performed with human ␣-thrombin at the indicated concentrations for 1 h at 37 °C in 20 mM KPO4, pH 7.5, 100 mM NaCl. All lanes had 40 ␮g of protein and lanes 7–9 included a final concentration of 4 M urea in the sample loading buffer. The arrows indicate monomeric (m) and dimeric (d) receptor species.


H6PAR1 (KY91) is similar to that of H6T7Ste2. In non-permeabilized cells, the median fluorescence intensity of KY91 increases 1.8-fold (Fig. 2A) and 1.5-fold (Fig. 2B) relative to control cells using the SFLLR-Ab and T7-Ab, respectively. In permeabilized cells, the median fluorescence intensity increases 3.4- and 3.8-fold relative to control cells, respectively. Thus, PAR1 is also distributed on both plasma membrane and intracellular membranes. Since the SFLLR and T7 epitopes are located in the N-terminal exodomain of the H6PAR1, the staining of intact yeast spheroplasts by the two antibodies also indicates that the receptor is in the proper orientation in the plasma membrane for a type III integral membrane protein. Co-Purification of G␣ Subunits with PAR1—Next, we examined the ability of co-expressed G␣ subunits to bind His6-tagged PAR1. The first G␣ subunit to be tested for binding to PAR1 was full-length Gi2. The yeast-expressed Gi2 comigrates with authentic Gi2 from platelet membranes at 38 kDa (Fig. 3). The yeast membranes containing H6PAR1 plus Gi2 subunit were passed over Ni-IDA columns and extensively washed with buffer containing 0.1% CHAPS detergent. Bound H6PAR1䡠Gi2

complexes were eluted with 100 mM imidazole and Western analysis performed with the SFLLR-Ab to detect the presence of H6PAR1. As shown at the bottom of Fig. 3, lane 4, H6PAR1 is eluted from the Ni-IDA resin by 100 mM imidazole and migrates as a 34 kDa band. Gi2 copurifies with H6PAR1 as detected by the AS-Ab (Fig. 3, lane 4). Gi2 is not retained on the Ni-IDA resin in the absence of co-expressed H6PAR1 consistent with specific Gi2/H6PAR1 binding occurring under these conditions (Fig. 3, lane 6). We determined that the yeast Gpa1 subunit does not copurify with H6PAR1. Since the genomic GPA1 allele gives low levels of Gpa1 protein expression, we overexpressed Gpa1 5–10-fold over background levels using a high copy number plasmid (Fig. 3, lane 14). As shown in Fig. 3, lane 15, even these high levels of Gpa1 do not bind to H6PAR1. Since the fulllength Gpa1 does not bind to H6PAR1 it was used as a null structural template in the subsequent chimera studies. We could then add back portions from authentic couplers such as Gi2 or G16 and test for gain of binding in order to delineate domain-specific functions within the G␣ subunits. The chimeric G␣ subunits were constructed using C- and N-terminal portions of mammalian Gi2, G16, and the endogenous yeast Gpa1: Gpa1-i2, Gpa1–16, and Gi2-gpa1 (Fig. 6). The Gpa1-i2 chimera comprises residues 1–329 of the yeast Gpa1 and residues 213– 355 of Gi2 (34). The Gpa1–16 chimera comprises residues 1–329 of Gpa1 joined to residues 220 –374 of G16. The reverse Gi2gpa1 chimera comprises residues 1–212 of the Gi2 joined to residues 330 – 472 of Gpa1. The chimeric junction (Fig. 6) is located at the highly conserved switch II region (25). To simplify interpretation of the Western data, we deleted the genomic GPA1 allele (Fig. 3, lane 16) and expressed the Gpa1 chimeras in the gpa1 null background. The chimeric G proteins, Gpa1-i2 and Gpa1–16, were expressed in yeast, and both co-purify with H6PAR1 (Fig. 3, lanes 10, 11, and 13, respectively). Therefore, the C-terminal third of Gi2 or G16 (48) is sufficient to confer binding to PAR1 although the majority of the chimera is Gpa1. The reverse chimera, Gi2-gpa1, which had been reported in earlier studies to be expressed in yeast (34) is not detected by the AS-Ab or Gpa1-Ab. However, the Gi2-gpa1 reverse chimera was found to couple efficiently to PAR1 by functional assays (see below). Thrombin Receptor Agonist Peptide (TRAP) Binding to PAR1-G Protein Complexes—Next, we ascertained whether the heterologously produced PAR1-G protein complexes could bind SFLLRN peptide ligand. We prepared a high affinity radioligand, 125I-56, that has an EC50 of 30 nM for the human platelet PAR1 (39). Membrane fractions containing H6PAR1-G protein complexes were bound to Ni-IDA beads and washed extensively to reduce nonspecific binding of the hydrophobic 125I-56 peptide ligand. As shown in Fig. 4, SFLLRN peptide displaces 125I-56 from H6PAR1/Gi2 and H6PAR1/Gi2-Gpa1 with IC50 values of 2–3 ␮M (Table II). The maximal amount of bound 125I-56 is in the range of 10 –13 pM (Table II). Cells expressing Gpa1 along with H6PAR1 gave an IC50 value of 54 ␮M for the SFLLRN ligand. Since the endogenous Gpa1 does not bind H6PAR1 under these conditions, this 22-fold poorer IC50 value probably reflects the low affinity state of PAR1 in the absence of bound G protein. For comparison, 10-fold shifts in KD were seen with ␣-factor peptide binding to Ste2 when GTP␥S was added to dissociate bound Gpa1/␤␥ (49). In a negative control experiment, NAc-TRAP peptide, which does not bind the receptor, could not displace 125I-56 from H6PAR1/Gi2 at concentrations as high as 250 ␮M. In a second negative control experiment using yeast extracts that lacked the thrombin receptor, H6T7Ste2 was overexpressed and membrane extracts bound to Ni-IDA beads as before. In this case, very low levels (0.2 pM) of


FIG. 2. Cellular distribution of PAR1 expressed in yeast. Flow cytometry was performed on yeast cells that were treated with Lyticase to remove the cell wall and fixed with 5% formaldehyde. Cells were stained with the anti-SFLLR and anti-T7 antibodies as indicated and the data represent 10,000 gated events. Cellular distribution of PAR1 or Ste2 was determined in the presence or absence of 0.1% Tween 20 which permeabilized the plasma membrane to antibody as described under “Experimental Procedures.” A, control strain (. . . . .) KY34 (harbors p112-H6T7Ste2⌬300) was permeabilized with 0.1% Tween 20; gray area, KY91 (harbors p112-H6(T7)PAR1 and pPGK-Gi2-gpa1); —, KY91 permeabilized with 0.1% Tween 20. B, control strain (. . . . .) KY27 (harbors p112-H6Ste2⌬300) was permeabilized with 0.1% Tween 20; gray area, KY91; —, KY91 permeabilized with 0.1% Tween 20. C, control strain (. . . . .) KY27 permeabilized with 0.1% Tween 20; gray area, KY34; —, KY34 permeabilized with 0.1% Tween 20.

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2632


FIG. 4. Displacement of high-affinity radioligand 125I-56, by PAR1 peptide ligands. Yeast membrane proteins were bound to NiIDA beads and washed with 0.1% CHAPS-containing buffer. ProteinNi-IDA beads (25 ␮l/assay) were incubated with 0.7– 0.9 nM 125I-56, for 30 min, 4 °C, in the presence of varying concentrations of TRAP peptide as described under “Experimental Procedures.” Membrane proteins were from strains: ●, KY91 (H6PAR1/Gi2-gpa1); f, KY92 (H6PAR1/ Gi2); Œ, KY64 (H6PAR1/Gpa1); , KY33 (H6T7Ste2⌬300/Gpa1); 䉬, KY92 (H6PAR1/Gi2) titrated with NAc-TRAP instead of TRAP. The nonspecific background binding of 125I-56 to Ni-IDA beads alone was subtracted from each data point. The binding curves were fit by nonlinear regression using KaleidaGraph and allowing IC50, Boundmax, Boundmin, and Hill coefficient, n, to float. The best values and standard errors for IC50, and Boundmax (pM) are listed in Table II. Each binding curve is the average of triplicate samples and is representative of three independent experiments. 125 I-56 bind to Ni-IDA-H6T7Ste2 and this level is unaffected by SFLLRN peptide (Fig. 4). Functional Coupling of PAR1-G Protein Complexes Expressed in Yeast—Now that we had detected binding between PAR1 and G␣ subunits, we tested whether the complexes would undergo nucleotide exchange (19, 41) in response to the SFLLRN agonist. We devised a GTP-␥-35S nucleotide exchange assay that was optimized for our His6-tagged PAR1. As shown in Fig. 5, addition of SFLLRN to the H6PAR1䡠Gpa1–16, H6PAR1䡠Gi2-gpa1, or H6PAR1䡠Gi2 complexes stimulate binding of GTP-␥-35S with EC50 values ranging from 1.8 to 4.4 ␮M (Table II). Addition of GDP displaces ⱖ96% of bound GTP-␥-35S from the SFLLRN-activated H6PAR1䡠Gi2 complex (Fig. 5C). Similar reductions are seen for the H6PAR1䡠Gi2-Gpa1 and H6PAR1䡠Gpa1–16 complexes (data not shown). There is no

SFLLRN-dependent binding of GTP-␥-35S to Gpa1 coexpressed with H6PAR1 (Fig. 5C), as expected since Gpa1 does not bind PAR1. Coupling by Gi2-gpa1 shows that the N-terminal twothirds of Gi2 provide sufficient functional interactions with PAR1, and that the C terminus of Gpa1 can substitute for Gi2 to provide the essential C-terminal coupling determinants. Conversely, the Gpa1–16 chimera couples to PAR1 thus demonstrating that the C terminus of G16 can also provide sufficient positive interactions to dominate the otherwise deficient interactions occurring at the N terminus of Gpa1 (48). Most surprisingly, PAR1 does not couple to the bound Gpa1-i2 chimera despite having the appropriate C-terminal ␤6/␣5 motif (Fig. 6). This is also a first indication that N-terminal regions of the G␣ subunit can supersede C-terminal regions in the control of G protein-PAR1 coupling. The ␣N helix is the prime candidate within the N-terminal two-thirds of the G␣ subunit for mediating coupling between the receptor and G protein. This ␣N helix is the site of Nterminal lipid modification of the G␣ subunit and is expected to orient the G protein heterotrimer in juxtaposition to the plasma membrane and the intracellular loops of the G proteincoupled receptors (25). Early support for this model was provided by Hamm et al. (50) who demonstrated that an 8 –23 Gt peptide blocked full-length Gt from binding rhodopsin. The first direct evidence for this topological model was provided by Neubig and colleagues (51) who detected cross-linking between intracellular loops from the ␣2-adrenergic receptor and the N-terminal 16 residues of the Gi/o subunit. In order to ascertain whether the ␣N-helix (residues 6 –30) of Gi2 may contain the N-terminal coupling determinants, we replaced Gi2 residues 1–34 with the corresponding residues from Gpa1 (residues 1– 42) to make the ␣N-i2 chimera. This chimera retains strong binding to H6PAR1 by Ni-IDA chromatography (Fig. 3, lane 8), but does not couple to PAR1 (Fig. 5B) unlike full-length Gi2. To our knowledge, this is the first report that receptor-G protein coupling determinants are located within the ␣N helix of a G␣ subunit and is contrary to the dogma that C-terminal G␣ regions are paramount for coupling interactions. In assessing the fidelity of coupling between our heterologously expressed PAR1/G␣ subunit pairs, we examined the analogous parameters derived from mammalian systems. The SFLLRN-dependent nucleotide exchange EC50 values which


FIG. 3. Purification of His6-tagged PAR1-G protein complexes by Ni-chelate chromatography. Top, Western blots (10% SDS-PAGE) of protein chromatographed on a 5-ml Ni-IDA column using antibodies directed against the C-tail of Gi2 (AS-Ab) or against whole Gpa1 (Gpa1-Ab) as described under “Experimental Procedures.” Loaded material is designated Ni⫺ and material eluted from the Ni-IDA column with 100 mM imidazole is designated Ni⫹. Bottom, anti-SFLLR Western blots of identical material loaded in corresponding top lanes. All lanes contained 40 ␮g of protein.


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TABLE II PAR1/G protein ligand binding and coupling parameters Individual 125I-56 ligand binding experiments are shown in Fig. 4. GTP-␥-35S binding experiments were conducted as described in the legend to Fig. 5. Radioligand

Receptor

G protein

SFLLRN IC50 or EC50

␮M

pM

I-56 125 I-56 125 I-56 ⫹ NAc-TRAPb 125 I-56 125 I-56 GTP-␥-35S GTP-␥-35S GTP-␥-35S GTP-␥-35S ⫹ GDPc GTP-␥-35S GTP-␥-35S GTP-␥-35S

PAR1 PAR1 PAR1 PAR1 Ste2 PAR1 PAR1 PAR1 PAR1 PAR1 PAR1 PAR1

Gi2-gpa1 Gi2 Gi2 Gpa1 Gpa1 Gi2-gpa1 Gpa1–16 Gi2 Gi2 Gpa1-i2 ␣N-i2 Gpa1

3⫾1 2⫾1 ⬎1 mM 54 ⫾ 30

13 ⫾ 1 10 ⫾ 4 10 ⫾ 1 11 ⫾ 1 0.2 ⫾ 0.1 5.0 ⫾ 1.0 4.3 ⫾ 0.8 2.0 ⫾ 0.2 0.07 ⬍0.5 ⬍0.5 ⬍0.3

125

a b c

Referred to as Boundmax in Figs. 4 and 5. Titration was performed with NAc-TRAP instead of SFLLRN and did not displace bound 10 ␮M GDP was included in reaction mixture.

⬎500 ⬎1000 ⬎1000 125

I-56.


we obtained for individual Gi2, Gpa1-G16, and Gi2-Gpa1 subunits coupled to PAR1 agree nicely with those values derived from platelets. Seiler et al. (19) obtained EC50 values of 1–3 ␮M for GTPase activation with platelet membranes. Since platelet extracts contain at least 9 G␣ subtypes (52) these EC50 values must be considered to be an aggregate measurement although inhibition with pertussis toxin indicated that the majority of the GTPase activity derives from Gi subunits. In the studies presented here, we have made the first quantitative measurements of coupling between isolated PAR1-G protein pairs. Sequestration of Yeast ␤␥ by G␣ Subunits—Receptor-bound heterotrimeric G proteins consist of GDP-G␣䡠␤␥ complexes. G␣ subunits do not support coupling to G protein-coupled receptors in the absence of ␤␥ subunits (53). Since the Gi2, Gi2-gpa1, and Gpa1-G16 chimeras in complex with PAR1 undergo high-affinity SFLLRN-dependent nucleotide exchange, this would indicate that the yeast ␤␥ dimer associates at least transiently with these G␣ subunits. Indeed, if null mutants are made by deleting either the yeast STE4 or STE18 genes which encode the yeast ␤ and ␥ subunits, respectively, one does not observe high affinity binding of ␣-factor peptide to the Ste2 receptor (49). The intersubunit contacts between G␣ and ␤ subunits are highly conserved between yeast and mammals (70% identity) and are well defined by x-ray structural analysis (25, 26). The major G␣ switch I-II interface buries 1800 Å2 of surface area with the side of the ␤ subunit (Fig. 6) and undergoes substantial conformational changes upon nucleotide exchange (25). A secondary interface between the top side of the ␤ subunit and the ␣N helix of G␣ provides an additional 900 Å2 of binding surface. To directly test for the ability of the hybrid mammalian G␣ subunits to bind the yeast ␤␥ dimer we employed a ␤␥ sequestration assay. An essential function of the yeast G␣ subunit, Gpa1, is to tightly sequester ␤␥ and thus prevent it from activating the MAP kinase scaffolding protein complex, Ste5䡠Ste20䡠Cdc24 (54, 55). Even minor leakage of free ␤␥ away from G␣ results in activation of the MAP kinase cascade and mitotic arrest (34). The growth arrest phenotype is dependent on the activity of the Far1 protein. The opposite phenotype occurs with our genetically altered yeast strain KY187 (far1⌬ lys2::FUS1-HIS3 his3⌬200 gpa1⌬). This strain has been engineered so that growth in histidine-deficient media is dependent on ␤␥ activation of the MAP kinase pathway which results in transcription of an integrated FUS1-HIS3 reporter gene (56). The growth arrest that would normally occur due to activation of the MAP kinase pathway is prevented by deletion of the

1.8 ⫾ 1.7 1.8 ⫾ 0.9 4.4 ⫾ 0.9

Maximal bounda

FIG. 5. SFLLRN-dependent GTP␥S binding to PAR1-G protein complexes. Ni-IDA-protein complexes were prepared from yeast membranes as described in the legend to Fig. 4 except that 15 ␮l of beads were used per assay. SFLLRN-dependent GTP␥S binding assays were performed in 200-␮l volumes with 1-h incubations at 30 °C in the presence of 0.5 nM GTP␥35S. The nonspecific background binding of GTP-␥-35S to Ni-IDA beads alone was subtracted from each data point. A: E, KY91 (H6PAR1/Gi2-gpa1) membranes. 〫, KY181 (H6PAR1/ Gpa1–16) membranes. B: ƒ, KY175 (H6PAR1/Gpa1-i2) membranes; f, KY321 (H6PAR1/␣N-i2) membranes. C: ‚, KY64 (H6PAR1/Gpa1) membranes; 䡺, KY92 (H6PAR1/Gi2) membranes. The downward arrow at the bottom right indicates displacement of maximally bound GTP-␥-35S from KY92 (H6PAR1/Gi2) membranes by 10 ␮M GDP (●). The EC50 and Boundmax values are listed in Table II. Each binding curve is the average of triplicate samples and show the data selected from one of five to seven independent experiments.

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FAR1 gene in KY187. Sequestration of ␤␥ by a G␣ subunit results in slow growth (long doubling time) due to failure to activate the MAP kinase pathway and transcription of the FUS1-HIS3 reporter gene. As shown in Table III, plasmidexpressed Gpa1 sequesters ␤␥ and strongly retards growth (⬎15 h doubling time) in the absence of histidine. Addition of histidine to the media bypasses the necessity for the MAP kinase-dependent transcription of the FUS1-HIS3 reporter and restores growth (3.9-h doubling time). Conversely, in the absence of Gpa1 (vector alone), the ␤␥ dimer freely activates the MAP kinase pathway, which results in the transcription of FUS1-HIS3. This causes a marked increase in growth (3.4-h doubling time) in histidine-deficient media. Expression of fulllength Gi2 in KY187 results in little or no sequestration of ␤␥ as determined by our growth assay (Table III). Another study (34) had previously demonstrated very weak sequestration of yeast ␤␥ by Gi2 in FAR1⫹ yeast strains using a mitotic arrest assay. In order to determine which domains of the G␣ subunit confer the ability to associate tightly with yeast ␤␥, the array of G␣ hybrids were tested in the KY187 yeast strain. The Gi2gpa1 chimera does not sequester ␤␥ indicating that the Nterminal two-thirds of Gi2 does not provide sufficient positive interactions with yeast ␤␥. Conversely, the Gpa1-i2 and ␣N-i2 chimeras provide full sequestration of ␤␥ (Table III). This clearly indicates that the positive interactions afforded by the ␣N helix are necessary for sequestration of yeast ␤␥ despite the remainder of the G␣ subunit being comprised of Gi2 including the switch I-II interface (Fig. 6). Surprisingly, unlike Gpa1-i2, the Gpa1–16 chimera does not sequester yeast ␤␥ irrespective of the 90% identity between Gpa1 and Gpa1–16 at positions that directly contact ␤␥ in both N-terminal and switch I-II regions. Therefore, negative interactions from the C-terminal third of the G␣ subunit may overcome positive interactions that exist in the N-terminal interface region. Together, these

TABLE III Sequestration of yeast ␤␥ by plasmid-expressed G␣ subunits inhibits growth in histidine-deficient media G␣ chimera

Doubling timea without histidine

Doubling time with histidine

h

h

Gpa1 Vector alone Gpa1-i2 Gi2-gpa1 Gpa1–16 Gi2 ␣N-i2

⬎15b (n ⫽ 5) 3.4 ⫾ 0.1 (n ⫽ 2) ⬎23b (n ⫽ 3) 3.3 ⫾ 0.1 (n ⫽ 3) 3.0 ⫾ 0.1 (n ⫽ 5) 3.7 ⫾ 0.3 (n ⫽ 3) ⬎16b (n ⫽ 4)

3.9 NDc 4.5 ND ND ND 3.6

Sequestration of ␤␥

Sequesters None Sequesters None None None Sequesters

a Doubling time (dt) in hours from growth of gpa1 far1 his3 FUS1HIS3 yeast KY187 carrying different G␣-expressing plasmids in histidine-deficient media as measured by absorbance (A600); dt ⫽ 0.3(t2-t1)/ (logA2-logA1). b Yeast culture growth over 2–3 days established only a lower limit of the doubling time. c ND, not determined.

data would imply that the forces holding together the heterotrimer are highly cooperative as suggested by x-ray structural analyses (25, 26). Dual Function of the G␣ Subunit ␣N Helix in Binding to ␤␥ and in Coupling to Receptor—The biochemical and genetic data presented here are the first evidence that the ␣N helix of the G␣ subunit plays a critical role in mediating proper coupling between G protein and receptor. The ␤␥-sequestration data demonstrate that binding determinants in the N-terminal ␣N helix are necessary but not sufficient for tight binding to the ␤␥ dimer. Based on the x-ray structure of G␤␥t (25), seven residues from the yeast Ste4 ␤ subunit would comprise the binding surface for interaction with six residues from the bottom face of the ␣N helix of G␣ (Fig. 6). Three of the seven Ste4 N-terminal interface residues are non-conserved relative to mammalian ␤1


FIG. 6. Domain-specific interactions between PAR1 and G␣ subunits. Composition of the hybrid G␣ subunits and their characteristics related to the heterotrimeric G protein structure (25). Labels on the G␣ subunit are as follows: ␣N is the N-terminal ␣-helix involved in receptor and ␤␥ coupling, ␣B domain is a unique Gpa1 domain of unknown function, SwII is part of the G␣ switch I-II interface that binds ␤␥ and undergoes nucleotide-dependent conformational changes, the ␤6/␣5 region is a C-terminal region involved in receptor coupling.

PAR1 Thrombin Receptor-G Protein Interactions (Leu 3 Asn, Phe 3 Lys, Phe 3 Lys). These substitutions could prevent association with non-Gpa1 subunits and thereby explain the inability of full-length Gi2 to sequester yeast ␤␥. Alanine scanning mutagenesis confirmed the importance of the cognate ␣N helix residues of Gt in binding mammalian ␤␥ (27). Point mutations of four out of six ␣N residues that contact ␤ were able to disrupt Gt binding to the ␤␥ dimer. In marked contrast, the alanine scan failed to detect deleterious effects on rhodopsin binding to Gt upon mutation of the interspersed surface-exposed residues of the ␣N helix. Likewise, in our studies here, the ␣N-i2 chimera bound tightly to PAR1 despite having several non-conserved substitutions at surface-exposed residues on the ␣N helix. The ␣N-i2 chimera did not couple to PAR1, however, indicating that ␣N helix provides fine determinants of G protein-receptor signaling and is not simply docking the G protein to the receptor-membrane interface.

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Acknowledgments—We are grateful to Martin Beinborn, Alan Kopin, and Maria Diverse for crucial advice and helpful discussions. We thank Amy Gresser for technical assistance, David Stone for antibodies, Susan DeSimone and Janet Kurjan for the G-protein plasmids, Tom Connolly for peptide 56, and Elaine Elion and members of her laboratory for yeast strains and plasmids.

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PROTEIN CHEMISTRY AND STRUCTURE: PAR1 Thrombin Receptor-G Protein Interactions: SEPARATION OF BINDING AND COUPLING DETERMINANTS IN THE Gα SUBUNIT Steven Swift, Paul J. Sheridan, Lidija Covic and Athan Kuliopulos

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J. Biol. Chem. 2000, 275:2627-2635. doi: 10.1074/jbc.275.4.2627