Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinasesстатья из журнала
Аннотация: G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2–14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation. G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2–14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation. A diverse array of extracellular stimuli transduce their signals through interaction with G protein-coupled receptors (GPCRs). 1The abbreviations used are: GPCRG protein-coupled receptorGRKG protein-coupled receptor kinaseRGSregulator of G protein signalingSST-14somatostatin-14SSTR2somatostatin receptor subtype 2β2ARβ2-adrenergic receptor.1The abbreviations used are: GPCRG protein-coupled receptorGRKG protein-coupled receptor kinaseRGSregulator of G protein signalingSST-14somatostatin-14SSTR2somatostatin receptor subtype 2β2ARβ2-adrenergic receptor. A critical process that occurs in most cells is the regulation of hormonal responsiveness, a phenomenon often termed desensitization. Whereas multiple mechanisms contribute to the regulation of GPCR function, G protein-coupled receptor kinases (GRKs) and arrestins play an important role in many cells (1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 2Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 3Perry S.J. Lefkowitz R.J. Trends Cell Biol. 2002; 12: 130-138Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). GRKs comprise a family of serine/threonine kinases that are uniquely able to associate with the agonist-occupied form of receptors (3Perry S.J. Lefkowitz R.J. Trends Cell Biol. 2002; 12: 130-138Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 4Stoffel R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar, 5Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). Signaling is terminated upon receptor phosphorylation and the subsequent binding of arrestins, effectively uncoupling receptor from G protein. Mammalian GRKs are classified into three subgroups according to their sequence homology: GRK1 and -7; GRK2 and -3; and GRK4, -5, and -6 (4Stoffel R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar, 5Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 6Penn R.B. Pronin A.N. Benovic J.L. Trends Cardiovasc. Med. 2000; 10: 81-89Crossref PubMed Scopus (185) Google Scholar). GRKs share a common structural organization that includes a moderately conserved (∼20% identity) NH2-terminal domain of ∼185 residues, a conserved (∼50% identity) central catalytic domain of ∼330 residues, and a poorly conserved COOH-terminal domain of ∼80–180 residues (4Stoffel R.H. Pitcher J.A. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar, 5Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar).An important feature of GRKs is their ability to specifically interact with activated receptors. Although the specific determinants required for this recognition are not well defined, a few studies have suggested a role for the GRK NH2-terminal region in receptor interaction. For example, site-specific antibodies directed against regions of the NH2-terminal region of GRK1 blocked phosphorylation of light-activated rhodopsin but had no effect on phosphorylation of a peptide substrate (7Palczewski K. Buczylko J. Lebioda L. Crabb J.W. Polans A.S. J. Biol. Chem. 1993; 268: 6004-6013Abstract Full Text PDF PubMed Google Scholar). In addition, NH2-terminal truncation mutants of GRK1 and GRK2 as well as point mutants targeting a conserved acidic residue in the NH2 terminus resulted in a greatly decreased ability of these GRKs to phosphorylate rhodopsin, whereas activity toward a peptide substrate was unaffected (8Yu Q.M. Cheng Z.J. Gan X.Q. Bao G.B. Li L. Pei G. J. Neurochem. 1999; 73: 1222-1227Crossref PubMed Scopus (30) Google Scholar). In addition to interaction with the receptor, previous studies implicating negatively charged phospholipids in GRK activation also have suggested a role for the NH2 terminus in phospholipid association (9Pitcher J.A. Fredericks Z.L. Stone C.W. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar).In the present study we attempted to further elucidate the role of the NH2 terminus of GRKs with respect to receptor interaction. We make use of an expression system whereby analysis of receptor-GRK association was facilitated through functional co-expression of these proteins in yeast cells. Saccharomyces cerevisiae utilizes GPCRs as well as heterotrimeric G proteins to regulate mating between two haploid yeast cells (10Kurjan J. Annu. Rev. Biochem. 1992; 61: 1097-1129Crossref PubMed Scopus (141) Google Scholar, 11Wittenberg C. Reed S.I. Curr. Opin. Cell Biol. 1996; 8: 223-230Crossref PubMed Scopus (41) Google Scholar, 12Banuett F. Microbiol. Mol. Biol. Rev. 1998; 62: 249-274Crossref PubMed Google Scholar). Mating pheromones bind to pheromone-specific GPCRs encoded by the STE2 and STE3 genes. Upon activation, these pheromone receptors promote the activation of the G protein, GPA1, which then activates a downstream signal transduction pathway. As a result, transcriptional activation of additional components of the mitogen-activated protein kinase cascade occurs, leading to cell cycle arrest. Previously, it has been demonstrated that mutations of certain components of the yeast pheromone response pathway eliminates the characteristic growth arrest response (13Price L.A. Kajkowski E.M. Hadcock J.R. Ozenberger B.A. Pausch M.P. Mol. Cell. Biol. 1995; 15: 6188-6195Crossref PubMed Scopus (122) Google Scholar). This in turn led to the development of a yeast-based bioassay using strains that were constructed to functionally express the rat somatostatin receptor subtype 2 (SSTR2) and consequently induce growth upon activation by somatostatin peptide. In our investigation, we coexpressed mammalian GRKs with the SSTR2 in yeast to determine any possible phenotypic effects of these kinases. Interestingly, our results revealed that co-expression of GRK2 or GRK5 resulted in loss of the agonist-dependent growth response observed with receptor alone. This bioassay was then used to select NH2-terminal mutants of GRK5 that are selectively deficient in inhibiting agonist-promoted growth. We demonstrate that several residues within the NH2-terminal region of GRK5 are important for phosphorylation of receptor substrates. In addition, we also demonstrate that this region plays a role in mediating the association of GRK5 with phospholipids. Thus, our studies have identified an NH2-terminal domain of GRK5 important for receptor phosphorylation and phospholipid interaction.EXPERIMENTAL PROCEDURESMaterials—Restriction endonucleases were from New England Biolabs and Roche Applied Science. Somatostatin-14 and α-factor peptide were from Bachem. SP-Sepharose was from Amersham Biosciences, whereas phosphatidylcholine (soybean type II-S) was from Sigma. Monoclonal antibodies (anti-GRK 4–6, clone A16/17) against the GRK5 COOH terminus were from Upstate Biotechnology. Horseradish peroxidase-conjugated anti-rabbit antibody was from Sigma. Plasmid miniprep, PCR purification, and gel extraction kits were from Qiagen, whereas ECL reagents were from Pierce. FuGENE™ transfection reagent was from Roche Applied Science and [γ-32P]ATP was from Invitrogen. BacPAC baculovirus expression system was from Clontech. Peptides corresponding to residues 1–14 of GRK5 (MELENIVANTVLLK) as well as a scrambled peptide (TILLKVAVNNELEM) were synthesized by the solid state Merrifield method on an Applied Biosystems automated synthesizer and purified by reverse-phase high performance liquid chromatography by the Kimmel Cancer Center Protein Facility.Plasmid Construction—The plasmid pMP222 was constructed by amplifying a fragment encoding the rat SSTR2 by PCR using pJH2 (13Price L.A. Kajkowski E.M. Hadcock J.R. Ozenberger B.A. Pausch M.P. Mol. Cell. Biol. 1995; 15: 6188-6195Crossref PubMed Scopus (122) Google Scholar) as template and synthetic oligonucleotides (MPO249, TCTCAAGCTTAAAAATGGAGATGAGCTCTGAG; MPO250, TCTCAGATCTTCAGATACTGGTTTGGAGG) that add a 5′ HindIII site followed by a yeast translation initiation site and a 3′ BglII site. The fragment was cut with HindIII and BglII and cloned into corresponding sites in pPGK (14Kang Y.S. Kane J. Kurjan J. Stadel J.M. Tipper D.J. Mol. Cell. Biol. 1990; 10: 2582-2590Crossref PubMed Google Scholar). The sequence of the SSTR2 fragment was confirmed by automated DNA sequencing.The plasmid pSST2-G418r was assembled from fragments amplified by PCR. Synthetic oligonucleotides (MPO68, ATAGAGCTCAGCTTACCGAATTTATCAATG; MPO72, ATAGGATCCACAAATGTATCATCATTATT) were used to amplify a 5′ fragment from yeast genomic DNA corresponding to nucleotides that encoded the first 238 amino acids of SST2 (15Dietzel C. Kurjan J. Mol. Cell. Biol. 1987; 7: 4169-4177Crossref PubMed Scopus (160) Google Scholar) while adding 5′ SacI and 3′ BamHI sites. A 3′ SST2 fragment containing sequence was amplified from yeast genomic DNA with oligonucleotides (MPO70, GCGAAGCTTGAGAGTCTTACTCATCT; MPO71, ATACTCGAGCATATGGAGTTTATTTGCTAAT) that added 5′ HindIII and 3′ XhoI sites. The coding sequence of the G418r gene was amplified from pRC-CMV (Invitrogen) using oligonucleotides (MPO37, ATGAGGATCCAAAAATGATTGAACAAGATGGATTG; MPO38, GAGAAGCTTTCAGAAGAACTCGTCAAGAAG) that added 5′ BamHI and 3′ HindIII sites and permitted in-frame fusion with the aminoterminal SST2 fragment. The fragments were cloned into pCRII (Invitrogen) and sequenced. The fragments were excised and assembled into pBKS (Stratagene) forming pSst2-G418r.The plasmid pFUS2-CAN1 was constructed from fragments amplified by PCR from yeast genomic DNA. Synthetic oligonucleotides (MPO128, AAAGGATCCGGTTTTCTTGTCTTTTTCTTAAG; MPO129, AAAGAGCTCGTTTCTAATAAACTAATCTTCAAG) were used to amplify a 5′ fragment of FUS2 while adding 5′ SacI and 3′ BamHI sites. A 3′ FUS2 fragment was amplified with oligonucleotides (MPO130, AAACTCGAGATGACTCTATAGCTACCGG; MPO131, AAAGGTACCCTCTTCATGTTTCACAATTTCAT) that added 5′ XhoI and 3′ KpnI sites. The coding sequence of the CAN1 gene was amplified using oligonucleotides (MPO110, AAAGGATCCAAAATGACAAATTCAAAAGAAGACGCC; MPO111, AAAGTCGACCTATGCTACAACATTCCAAAATTTGTC) that added 5′ BamHI and 3′ SalI sites. The fragments were cloned into pCRII (Invitrogen) and sequenced. The fragments were excised and assembled into pRS306 (16Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) forming pFUS2-CAN1. pFUS2-CAN1 was linearized with XbaI prior to transformation.PCR Mutagenesis—Full-length human GRK5 in pcDNA3 was used as template for PCR amplification using T7 (sense) and 5′-CCAAGCGCTTGCAGGCA-3′ (antisense, encoding residues 213–218 of GRK5). To increase the frequency of random mutations, a mutagenic mixture of dNTPs containing 8 mm dCTP was used in conjunction with a final concentration of 5 mm MnCl2 in PCR. Amplification involved denaturing template DNA at 94 °C for 1 min, annealing for 45 s at 55 °C, and extension for 1.5 min at 72 °C for 30 cycles followed by a final extension for 5 min at 72 °C. The resulting PCR products from 10 separate reactions were then pooled and purified.Construction of GRK5 Yeast Expression Vectors—Wild type human GRK5 in pcDNA3 was digested with BamHI and XbaI and subcloned into the yeast expression vector Ycplac111, which includes a GAL1/10 promoter. Ycplac111-GRK5 was digested at 2 internal restriction sites, BamHI and Bsu36I, to excise a 462-base pair open reading frame fragment (encoding residues 1–154) of GRK5. Randomly mutagenized GRK5 PCR products were digested with BamHI and Bsu36I and ligated into previously digested Ycplac111-GRK5 to reconstruct full-length GRK5 cDNAs.Construction of MPY576fc—Haploid S. cerevisiae strains YPH499 (MATaura3-52 leu2Δ1 his3Δ200 lys2-801 ade2-101 trp1Δ63, Stratagene) and MMOY11 (MATα ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 can1-100 Ole+) (17McCammon M.T. Veenhuis M. Trapp S.B. Goodman J.M. Bacteriology. 1990; 172: 5816-5827Crossref PubMed Google Scholar) were crossed, the resulting zygotes were identified microscopically and cultured on YPD plates. The diploid cells were induced to sporulate on appropriate media and the tetrads were dissected on YPD plates. Four spore tetrads were assessed for the presence of required nutrient markers. MPY566 (MATaura3 leu2 his3 lys2-801 ade2 trp1 can1-100) was used for further strain construction. Standard yeast media and culture conditions were employed (18Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2532) Google Scholar).MPY566 was modified by sequential deletion of several genes in the mating signal transduction pathway leading to yeast strains that produce a sensitive growth-based readout of GPCR activation. DNA mediated transformation of S. cerevisiae was carried out using the lithium acetate method (19St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1991; 20: 1425Google Scholar). The far1 gene was deleted using the far1ΔLYS2 construct in pLP80 (14Kang Y.S. Kane J. Kurjan J. Stadel J.M. Tipper D.J. Mol. Cell. Biol. 1990; 10: 2582-2590Crossref PubMed Google Scholar). The SST2 gene was inactivated by replacement with the SacI-XhoI fragment in pSST2-G418r, a construct that permits expression of a Sst2-G418r fusion protein, by selecting for G418 resistance on plates containing α-mating factor. The FUS1 gene was replaced with the FUS1-HIS3 allele in pSL1497 (20Stevenson B.J. Rhodes N. Errede B. Sprague G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar). Finally, the FUS2 gene was modified with pFUS2-CAN1 using the pop-in/pop-out replacement procedure (21Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1098) Google Scholar). FUS2 coding sequences were replaced with those of CAN1, thus placing CAN1 expression under control of the pheromone inducible FUS2 promoter. The structures of the modified loci in MPY576fc were confirmed by PCR analysis. As a result of the modifications described above, MPY576fc is capable of agonist-induced vegetative growth on selective media lacking histidine, G418 resistance, and sensitivity to the toxic arginine analog, canavanine.Bioassay—Cultures of the yeast strain containing the SSTR2 alone (MPY576fc(pMP222)), or SSTR2 plus GRK5 (MPY576fc(pMP222, Ycplac111-GRK5)) were grown overnight in synthetic complete media containing glucose (2%) and lacking uracil (to select for receptor alone), or containing galactose (2%) and lacking uracil and leucine (to select for receptor and GRK), then centrifuged for 10 min at 1000 × g. Pelleted cells were resuspended in 1 ml of sterile water and subsequently diluted 1000-fold. Diluted cells (0.3 ml) were then spread on agar plates lacking uracil and histidine or lacking uracil, leucine, and histidine. Plates also contained 4 mm 3-amino-1,2,4-triazole to inhibit background growth. Sixty pmol of somatostatin-14 peptide (SST-14) in a total volume of 5 μl was then spotted in the center of the plate and allowed to dry. Plates were incubated for ∼48 h until growth appeared on and around the point of agonist application. Analogous experiments using α-factor peptide (5 μl of 2 mg/ml) were performed using similar methods. Overnight cultures were grown in selective media containing either glucose or galactose, and cells were then pelleted, washed, and plated on agar media lacking uracil, leucine, and histidine in the presence of 3-amino-1,2,4-triazole.Selection Assays to Identify Mutants—Log phase cultures of strain MPY576fc(pMP222) containing the SSTR2 were grown in raffinose (1%) selection media lacking uracil and subsequently made competent by incubating cells with 0.1 m lithium acetate, 10 mm Tris-HCl, pH 8.0, 20 mm EDTA. 10 μl of ligation reactions generated from mutagenic PCR plus carrier DNA were added to the competent cells and then incubated at 30 °C for 1 h followed by heat shock treatment for 5 min at 42 °C in the presence of Me2SO. Cell suspensions were then plated on agar containing galactose and lacking uracil, leucine, and histidine (+4 mm 3-amino-1,2,4-triazole). 180 nmol of SST-14 peptide were added to the plates and incubated for 7 days. Colonies were then patched onto plates containing galactose and lacking uracil, leucine, and histidine and incubated in the presence or absence of SST-14 for 3–5 days.Expression of GRK5 in Yeast—GRK5 expression was confirmed by extraction of protein from yeast cells followed by Western blotting. Briefly, cultures of yeast strain MPY576fc(pMP222, Ycplac111-GRK5) containing SSTR2 and GRK5 expression vectors were grown to stationary phase in selective raffinose (1%) media, then washed and diluted 1:10 into selective galactose media. Cultures (10 ml) were grown to log phase to an A600 of ∼1.0, pelleted by centrifugation, and washed twice in 1 ml of buffer A (20 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 1 mm EDTA, 100 mm NaCl, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 40 μg/ml phenylmethylsulfonyl fluoride). Pelleted cells were resuspended in 100 μl of buffer A and 50 μl of this suspension was then diluted with an equal volume of SDS sample buffer and boiled for 10 min. Equal amounts of total protein were loaded on a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently electroblotted onto nitrocellulose membranes. Membranes were blocked for 30 min in 5% nonfat dry milk in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20 (TBST), then incubated for 1 h with GRK5 monoclonal antibody diluted 1:5000 in TBST + 5% dry milk. Membranes were washed 3 times in TBST, incubated for 1 h with peroxidase-labeled goat anti-mouse antibody (1:2000) in TBST + 5% dry milk, washed, and developed using ECL chemiluminescence reagent.Recovery of GRK5 Plasmids and Retransformation—Total DNA from individual isolates was extracted from yeast cells. Briefly, cells were grown overnight in selection media containing 1% yeast extract, 2% peptone, and 2% glucose. Cells were then centrifuged for 5 min at 1000 × g and resuspended in a final volume of 1 ml of 1 m sorbitol, pH 7.5, 0.1 m EDTA, 0.5 mg/ml zymolyase 60,000. Cells were incubated for 1 h at 37 °C followed by centrifugation and resuspension in 0.5 ml of 50 mm Tris-HCl, pH 7.4, 20 mm EDTA, 1% SDS. Cell suspensions were incubated at 65 °C for 30 min followed by the addition of 0.2 ml of potassium acetate, incubation on ice for 1 h, and addition of 1 volume of isopropyl alcohol. Samples were then incubated for 5 min at room temperature, centrifuged, and the pellets washed in 70% ethanol, airdried, and resuspended in 100 μl of 10 mm Tris-HCl, pH 7.4, 1 mm EDTA. Genomic DNA was transformed into the competent bacterial strain MC1066 that specifically selects for the Leu marker of the GRK5-containing plasmid. DNA was prepared from individual colonies and digested with BamHI/Bsu36I to verify the presence of plasmids containing GRK5. Yeast strain MPY576fc(pMp222) was then re-transformed with GRK5 cDNA-containing plasmids amplified in bacteria and colonies were re-tested in patch test assays for growth in the presence or absence of agonist. Plasmids were sequenced using the dideoxy chain termination method to identify mutations.Patch Tests—Re-transformed candidate clones were patched onto galactose-agar plates lacking uracil, leucine, and histidine in the presence and absence of SST-14. After ∼5 days, patches were qualitatively evaluated for growth compared with positive and negative controls (strains containing wild type GRK5 or SSTR2 alone, respectively).Transfection of COS-1 Cells—Expression plasmids for GRK5 were constructed as previously described (22Kunapuli P. Benovic J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5588-5592Crossref PubMed Scopus (117) Google Scholar, 23Pronin A.N. Carman C.V. Benovic J.L. J. Biol. Chem. 1998; 273: 31510-31518Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). COS-1 cells were grown to 80–90% confluence in 100-mm dishes in a humidified atmosphere containing 5% CO2, 95% air in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were transfected with 5 μg of DNA/dish using FuGENE™ as per the manufacturer's instructions. Cells were harvested 48 h after transfection and GRKs were partially purified by chromatography on SP-Sepharose as previously described (24Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Partially purified GRK samples typically contained 10–30 μg of GRK5/mg of protein.Purification of the β2-Adrenergic Receptor—The β2AR used in these experiments was modified with a cleavable signal sequence and FLAG epitope at the amino terminus, and a six histidine tag at the carboxyl terminus as previously described (25Kobilka B.K. Anal. Biochem. 1995; 231: 269-271Crossref PubMed Scopus (146) Google Scholar). Receptor was expressed in Sf9 cells, solubilized in 100 mm NaCl, 20 mm Tris-HCl, pH 7.5, 1% dodecylmaltoside, and 1 μm alprenolol, and purified by FLAG antibody chromatography (25Kobilka B.K. Anal. Biochem. 1995; 231: 269-271Crossref PubMed Scopus (146) Google Scholar).Purification of GRK5 and Substrate Phosphorylation—Wild type and mutant GRK5 were overexpressed and purified from Sf9 cells as described (26Kunapuli P. Onorato J.J. Hosey M.M. Benovic J.L. J. Biol. Chem. 1994; 269: 1099-1105Abstract Full Text PDF PubMed Google Scholar) and the purity was determined by Coomassie Blue staining. Protein concentration was determined by the dye binding assay (BioRad) using bovine serum albumin as standard. Purified GRK5 was assayed by incubating 20–100 nm kinase with various concentrations of rod outer segment membranes (1–10 μm rhodopsin), β2AR (50 nm), or tubulin (0.1–5 μm) in 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 0.1 mm [γ-32P]ATP (∼1000 cpm/pmol) in a final volume of 20 μl. The β2AR incubations also contained 0.85 mg/ml soybean phosphatidylcholine and were in the presence or absence of 50 μm isoproterenol. Samples were incubated for 1–60 min at 30 °C in room light, quenched with SDS buffer, and electrophoresed on a 10% polyacrylamide gel. Gels were stained with Coomassie Blue, dried, and autoradiographed. 32P-Labeled proteins were excised and counted to determine picomole of phosphate transferred.Autophosphorylation of Purified GRK5—Autophosphorylation reactions contained 4 pmol of purified wild type or mutant GRK5 in 20 μl of 20 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 0.1 mm [γ-32P]ATP (∼1000 cpm/pmol). Reactions were incubated for 1–30 min at 30 °C and quenched with SDS sample buffer. Samples were electrophoresed and the 32P-labeled proteins were excised and counted. Autophosphorylation reactions performed in the presence of phospholipids contained 0.85 mg/ml soybean phosphatidylcholine vesicles.GRK5 Binding to Phospholipids—Phospholipid vesicles were prepared by sonicating 85 mg of crude soybean phosphatidylcholine in 5 ml of buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.1 mm EDTA) on ice 4 times for 20 s. Phospholipid association with GRK5 was analyzed by incubating 80 ng (20 nm) of purified or partially purified GRK5 in the presence or absence of the indicated amount of phospholipid in 60 μl of buffer (20 mm Tris-HCl, pH 8.0, 2 mm MgCl2, 100 mm NaCl, 0.015% Triton X-100) for 5 min at 30 °C. Reactions were pelleted at 200,000 × g for 15 min and pellet and supernatant fractions were solubilized in SDS sample buffer. Equal aliquots of each fraction were electrophoresed, transferred to nitrocellulose, and visualized by immunoblotting using mouse monoclonal anti-GRK5 antibodies. Optical density of developed bands was assessed by densitometry and the amount of GRK5 bound to lipid was expressed as a percentage of the total.RESULTS AND DISCUSSIONDevelopment of a Yeast Bioassay to Analyze GRK Interaction with GPCRs—The mating pheromone pathway in the budding yeast S. cerevisiae utilizes a GPCR-heterotrimeric G protein complex to regulate mating between haploid cells. During the mating process, pheromone peptide binds to GPCRs encoded by the STE2 and STE3 genes that correspond to the MATa and MATα mating types, respectively. Subsequently, a signal transduction cascade is initiated that activates mitogen-activated protein kinase homologues encoded by the FUS1 and KSS1 genes, as well as regulatory protein kinases encoded by the STE7 and STE11 genes. Under normal conditions, the activation of this pathway results in cell cycle arrest mediated by the protein product of the FAR1 gene (10Kurjan J. Annu. Rev. Biochem. 1992; 61: 1097-1129Crossref PubMed Scopus (141) Google Scholar, 11Wittenberg C. Reed S.I. Curr. Opin. Cell Biol. 1996; 8: 223-230Crossref PubMed Scopus (41) Google Scholar, 12Banuett F. Microbiol. Mol. Biol. Rev. 1998; 62: 249-274Crossref PubMed Google Scholar). However, by introducing specific genetic mutations into a given strain, this response can be eliminated. When FAR1 is deleted, cell growth is permitted to continue in the presence of the activated pheromone pathway. Based on this concept, a novel expression system was developed that allows yeast cells expressing the mammalian somatostatin receptor subtype 2 to couple to the pheromone pathway in the presence of the peptide agonist SST-14 (13Price L.A. Kajkowski E.M. Hadcock J.R. Ozenberger B.A. Pausch M.P. Mol. Cell. Biol. 1995; 15: 6188-6195Crossref PubMed Scopus (122) Google Scholar). Activation of this pathway can then be used in a growth selection by placing a HIS3 reporter into transcriptional elements of the FUS1 gene. Expression of the His3 protein is thus placed under control of the pheromone response cascade such that binding of SST-14 and subsequent activation of the pathway induces the FUS1 promoter. His3 protein expression then permits auxotrophic growth of yeast cells on media lacking histidine. In accordance with this observation, an appropriate strain was constructed (MPY576fc) that functionally expresses the SSTR2 and exhibits agonist-dependent growth upon exposure to somatostatin (13Price L.A. Kajkowski E.M. Hadcock J.R. Ozenberger B.A. Pausch M.P. Mol. Cell. Biol. 1995; 15: 6188-6195Crossref PubMed Scopus (122) Google Sch
Год издания: 2003
Авторы: Beth Noble, Lorena A. Kallal, Mark H. Pausch, Jeffrey Benovic
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Receptor Mechanisms and Signaling, Protein Kinase Regulation and GTPase Signaling, Monoclonal and Polyclonal Antibodies Research
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 278
Выпуск: 48
Страницы: 47466–47476