A Gain-of-function Polymorphism in a G-protein Coupling Domain of the Human β1-Adrenergic Receptor

Abstract

The β1-adrenergic receptor (β1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human β1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0.74, respectively), the former previously considered as the human wild-type β1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels weremarkedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5′-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gsand increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5′-(β,γ-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (R H = 26%). Taken together, these data indicate that this polymorphic variation of the human β1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of β1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic βAR agonists and antagonists in cardiovascular and other diseases. The β1-adrenergic receptor (β1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human β1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0.74, respectively), the former previously considered as the human wild-type β1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 ± 1.2 versus 6.1 ± 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels weremarkedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 ± 6.1 versus 20.9 ± 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5′-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gsand increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5′-(β,γ-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (R H = 26%). Taken together, these data indicate that this polymorphic variation of the human β1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of β1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic βAR agonists and antagonists in cardiovascular and other diseases. The β1-adrenergic receptor (β1AR) 1The abbreviations used are: βAR, β-adrenergic receptor; Arg-389, β1AR polymorphism with Arg at amino acid 389; Gly-389, β1AR polymorphism with Gly at amino acid 389; 125I-CYP, 125I-labeled cyanopindolol; Gpp(NH)p, guanosine 5′-(β,γ-imido)triphosphate; GTPγS, guanosine 5′-O-(thiotriphosphate); PCR, polymerase chain reactionis a member of the adrenergic family of G-protein-coupled receptors, with epinephrine and norepinephrine being endogenous agonists. Like other members of the G-protein-coupled receptor superfamily, the amino terminus is extracellular, the protein is predicted to traverse the cell membrane seven times, and the carboxyl terminus is intracellular. In the adrenergic receptor family, agonists bind in a pocket formed by the transmembrane-spanning domains, and G-protein binding and activation occur at intracellular domains of the loops and tail, typically near the membrane (1Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (1008) Google Scholar). β1ARs couple to the stimulatory G-protein, Gs, activating adenylyl cyclase, as well as to non-cAMP pathways such as the activation of ion channels (2Yatani A. Brown A.M. Science. 1989; 245: 71-74Crossref PubMed Scopus (246) Google Scholar). β1ARs are expressed on a number of cell types including cardiomyocytes where they serve to increase cardiac inotropy and chronotropy, adipocytes where they mediate lypolysis, and juxtaglomerular cells of the kidney where they regulate renin secretion. It has been known for decades that these responses, as well as those of the β2AR, are somewhat variable in the human population (3Liggett S.B. Shah S.D. Cryer P.E. Am. J. Physiol. 1989; 256: E600-E609PubMed Google Scholar, 4Insel P.A. N. Engl. J. Med. 1996; 334: 580-585Crossref PubMed Scopus (180) Google Scholar). Indeed, we have recently found and characterized several β2AR polymorphisms that have altered signaling phenotypes compared with wild-type receptor (5Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar, 6Green S. Turki J. Innis M. Liggett S.B. Biochemistry. 1994; 33: 9414-9419Crossref PubMed Scopus (718) Google Scholar, 7Green S.A. Turki J. Bejarano P. Hall I.P. Liggett S.B. Am. J. Respir. Cell Mol. Biol. 1995; 13: 25-33Crossref PubMed Scopus (420) Google Scholar). This finding has prompted us to examine the β1AR coding sequence for variability in the human population. Here we report a common single nucleotide polymorphism resulting in a Gly to Arg switch at intracellular amino acid 389, within a region important for G-protein coupling. The resulting phenotype of the Arg-389 receptor is one of enhanced receptor-Gs interaction, functionally manifested as increased activation of the adenylyl cyclase effector. The wild-type sequence of the intronless human β1AR gene is considered that reported by Frielle et al. (8Frielle T. Collins S. Daniel K.W. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7920-7924Crossref PubMed Scopus (567) Google Scholar) (GenBankTMaccession numberJ03019). In the current report the adenine of the initiator ATG of the coding block is designated as nucleotide 1, and amino acid 1 is the encoded methionine. The receptor consists of 477 amino acids. Genomic DNA was extracted from blood or banked tissue from normal individuals by standard techniques (9Jones A.S. Nature. 1963; 199: 280-282Crossref PubMed Scopus (38) Google Scholar). The study was approved by the University of Cincinnati Institutional Review Board. PCR reactions were established to produce overlapping products spanning 1131 base pairs of the coding region starting from nucleotide 300 of the second transmembrane-spanning domain past the stop codon into the 3′-untranslated region. A consistent deviation from the published sequence was noted at nucleotide 951 where guanine was found in all subjects instead of adenine and likely represents a sequencing error in the original report. Both codons encode for Leu at amino acid 317. At nucleotide 1165, a guanine (“wild-type”) or cytosine was found (Fig. 1 A), altering the encoded amino acid from Gly to Arg at amino acid position 389. The primers used to amplify the 488-base pair fragment where this polymorphism at position 1165 was detected were: 5′-CAGGAAACAGCTATGACCACTGGAGCCGCCTCTTCGTCTTCTTCAACTG-3′ (sense) and 5′-TGGGCTTCGAGTTCACCTGCTATC-3′ (antisense). The sense primer contains a 5′ M13 forward primer sequence, so that dye primer chemistry using the universal primer could be utilized in the sequencing reactions. PCRs consisted of ∼500 ng of DNA, each dNTP at 62.5 nm, 100 pmol of each primer, 0.5 μl of Pwo polymerase (Roche Molecular Biochemicals), 10% of the supplied buffer, and 5% Me2SO in a final volume of 50 μl. Cycling conditions started at 98 °C for 5 min, followed by 98 °C for 45 s, 60 °C for 1 min, and 72 °C for 1 min for 35 cycles, and a final extension of 72 °C for 7 min in a Perkin-Elmer 9600 thermocycler. Automated sequencing was carried out with an Applied Biosystems sequencer using dye primer chemistry. At nucleotide 1165, the G to C transition results in a loss of a BsmF1 restriction endonuclease site. Additional samples were thus studied with the polymorphism identified by the absence or presence of digestion of the aforementioned PCR product with BsmF1 (Fig.1 B). Site-directed mutagenesis was carried out by methods previously described (5Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar) to mimic the Arg-389 polymorphism. Arg-389 and Gly-389 β1AR cDNAs were subcloned into the mammalian expression vector pBC12BI (10Cullen B. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (769) Google Scholar). CHW-1102 fibroblasts (CHW cells) were permanently transfected with the Gly-389 and Arg-389 β1AR constructs by calcium phosphate precipitation as described previously (11Green S.A. Holt B.D. Liggett S.B. Mol. Pharmacol. 1992; 41: 889-893PubMed Google Scholar). Positive clones were selected based upon resistance to 300 μg/ml G418. Cultures were maintained at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 80 μg/ml G418. COS-7 cells were transiently transfected with the β1AR constructs and rat Gαs in pCDNA1 for [35S]GTPγS binding studies. These transient transfections were performed by the DEAE-dextran/chloroquine method as described previously (12Green S.A. Spasoff A.P. Coleman R.A. Johnson M. Liggett S.B. J. Biol. Chem. 1996; 271: 24029-24035Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Confluent monolayers of CHW cells were washed three times with phosphate-buffered saline, lysed in a hypotonic buffer (5 mm Tris, 2 mm EDTA, pH 7.4), detached by scraping with a rubber policeman, and centrifuged at 42,000 ×g for 10 min. Crude membranes were resuspended in buffer (75 mm Tris, 12.5 mm MgCl2, 2 mm EDTA, pH 7.4) and saturation binding experiments performed with 125I-CYP as described (12Green S.A. Spasoff A.P. Coleman R.A. Johnson M. Liggett S.B. J. Biol. Chem. 1996; 271: 24029-24035Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), with nonspecific binding defined by co-incubation with 1 μm propranolol. Reactions for these and other radioligand experiments were terminated by dilution and vacuum filtration over glass fiber filters. One-site agonist competition binding studies were carried out with 40 pm125I-CYP, 100 μm GTP, and varying concentrations of isoproterenol, epinephrine, or norepinephrine for 2 h at 25 °C. To assess high and low affinity agonist receptor binding, membranes were prepared as described above, except two additional centrifugations were included before the addition of reaction buffer to assure the removal of endogenous GTP. Membranes were incubated with 40 pm125I-CYP and 18 concentrations of isoproterenol in the presence or absence of the nonhydrolyzable GTP analog Gpp(NH)p at 100 μm for 1 h at 37 °C. Competition data were fit to one-site and two-site models by an iterative least squares technique as described previously (13Green S. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar). A two-site model was considered valid if by F-test the fit was statistically better (p < 0.05) than that obtained with a one-site model. Membranes were incubated with 30 mm Tris, pH 7.4, 2.0 mmMgCl2, 0.8 mm EDTA, 120 μm ATP, 60 μm GTP, 2.8 mm phosphoenolpyruvate, 2.2 μg of myokinase, 100 μm cAMP, and 1 μCi of [α-32P]ATP for 30 min at 37 °C as described (11Green S.A. Holt B.D. Liggett S.B. Mol. Pharmacol. 1992; 41: 889-893PubMed Google Scholar). [32P]cAMP was separated from [α-32P]ATP by chromatography over alumina columns. A [3H]cAMP standard included in the stop buffer accounted for individual column recovery. Activities were determined in the presence of vehicle (basal), the indicated concentrations of agonists, or 100 μm forskolin. Transiently transfected COS cells were washed three times with phosphate-buffered saline, lysed in hypotonic buffer (5 mm HEPES, 1 mm EDTA, pH 8), detached by scraping with a rubber policeman, and centrifuged for 10 min at 42,000 × g. Pellets were resuspended in the same buffer and centrifuged two additional times. [35S]GTPγS binding was carried out by a modification of the method of Befort et al. (14Befort K. Tabbara L. Kieffer B.L. Neurochem. Res. 1996; 21: 1301-1307Crossref PubMed Scopus (41) Google Scholar). The reaction consisted of membranes in 10 mm HEPES, pH 7.40, 5 mmMgCl2, 100 mm NaCl, 1 mm EDTA buffer, with 1.0 μm GDP and 180 pm[35S]GTPγS in the presence of water (basal), 100 μm unlabeled GTPγS (to define nonspecific binding), or 10 μm isoproterenol for 2.5 h at 30 °C. Unbound [35S]GTPγS was separated using vacuum filtration over glass fiber filters that were washed twice with 10 mm Tris concentrations were determined by the acid method Biochem. PubMed Scopus Google Scholar). was carried out using were by or as with considered < are as ± of the indicated number of results from DNA from 30 individuals from a defined cohort in the β1AR region the coding block from the second domain to the amino acid in the carboxyl terminus of the in one (Fig. at nucleotide 1165, where guanine or cytosine could be resulting in a Gly or Arg at amino acid 389. Using BsmF1 restriction additional at this locus was carried out for a of 50 the wild-type human β1AR had previously been reported (8Frielle T. Collins S. Daniel K.W. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7920-7924Crossref PubMed Scopus (567) Google Scholar) as Gly at amino acid 389, we found a higher of Arg in normal population. The frequencies were for Arg and 0.26 for To the Arg in this position a phenotype compared with the Gly-389 receptor we (11Green S.A. Holt B.D. Liggett S.B. Mol. Pharmacol. 1992; 41: 889-893PubMed Google Scholar) and G. M. Mol. Pharmacol. 1996; Google Scholar) had previously site-directed mutagenesis was carried out to mimic Arg-389, and the two cDNAs were subcloned into mammalian expression The final constructs were except for the single nucleotide These constructs were used to of transfected CHW βAR studies were carried out on several from each at matched levels of receptor binding studies binding for Competition binding studies with isoproterenol, epinephrine, and norepinephrine in the presence of GTP differences the two receptors in binding for these binding of the two polymorphic ± ± ± ± ± ± ± ± were carried out in the presence of 100 μm GTP. in a were carried out in the presence of 100 μm GTP. and adenylyl cyclase activities were determined using membranes from Gly-389 at ± and Arg-389 at ± (Fig. 2 basal activities were slightly higher with the Arg-389 receptor than with the Gly-389 receptor (10.7 ± 1.2 versus 6.1 ± 0.4 = < The were with agonist isoproterenol-stimulated activities were higher with the Arg-389 receptor than with the Gly-389 receptor (63.3 ± 20.9 ± 2.0 = < the as over basal the Arg-389 receptor displayed a of adenylyl cyclase than the Gly-389 receptor ± versus ± In of adenylyl cyclase by was not the ± versus 120 ± 10 pmol/min/mg). the data to the of of adenylyl cyclase by a agonist by the Arg-389 than the Gly-389 (Fig. 2 B). The for isoproterenol of adenylyl cyclase for Gly-389 and Arg-389 receptors were not ± 5 ± 5 from an additional of studies carried out on other at higher expression levels are in C and was ± and ± for Gly-389 and Arg-389, the maximal of by agonist was with the Arg-389 These differences in adenylyl cyclase the two polymorphic receptors were also in to epinephrine and norepinephrine not To the basis of these differences the two receptors, studies were to assess receptor, and with [35S]GTPγS binding experiments and agonist competition studies in the absence and presence of [35S]GTPγS binding, we found that were obtained with high levels of transient receptor expression in COS-7 were with of these studies are in isoproterenol-stimulated [35S]GTPγS binding was found to be in membranes the Arg-389 versus the Gly-389 receptor, consistent with the adenylyl cyclase also enhanced with the Arg-389 A well of agonist binding to receptors such as the β2AR is the increase in high affinity binding of A. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. P. D. Mol. Pharmacol. 1992; 41: Google Scholar, P. S. T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). we have previously reported (13Green S. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar) that in competition studies carried out in the absence of Gpp(NH)p, accumulation of a β1AR high affinity is not This is likely of the of the high and low affinity consistent with signal the Arg-389 receptor in have enhanced high affinity binding, this receptor has increased functional coupling to competition studies were thus carried out in in the absence and presence of Gpp(NH)p, with washed membranes from CHW cells each These results are in and the in the absence of a guanine nucleotide from the Gly-389 experiments were ± and were resolved statistically to a one-site model. Gpp(NH)p had on agonist competition with the Gly-389 receptor (Fig. In the obtained with the Arg-389 receptor in the absence of Gpp(NH)p had of ± and the data were resolved with a two-site model < The of receptors in the high affinity with the Arg-389 receptor was ± (Fig. and with this receptor the presence of Gpp(NH)p in the reaction in the with consistent with a single low affinity agonist of agonist competition binding studies in the absence and presence of Gpp(NH)p with the Gly-389 and Arg-389 ± ± ± ± ± Gly-389 receptor could not be fit to a two-site model with than a one-site model for and thus high and low affinity binding were not of receptors in high affinity not in a The Gly-389 receptor could not be fit to a two-site model with than a one-site model for and thus high and low affinity binding were not of receptors in high affinity not These in the normal human population the presence of two β1AR genetic with differences in functional of and of the that a number of studies have been published using the Gly-389 receptor (13Green S. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar, T. Daniel K.W. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. U. S. A. PubMed Scopus Google Scholar), we to to this receptor as the human the Arg to be This is to the that has been with the common β2AR polymorphisms (5Green S.A. Cole G. Jacinto M. Innis M. Liggett S.B. J. Biol. Chem. 1993; 268: 23116-23121Abstract Full Text PDF PubMed Google Scholar, 6Green S. Turki J. Innis M. Liggett S.B. Biochemistry. 1994; 33: 9414-9419Crossref PubMed Scopus (718) Google Scholar). The of the variability is amino from the seventh transmembrane-spanning in the intracellular of the tail to the (Fig. This region is to as the intracellular in this report be the of the cytoplasmic with β2AR M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Caron M.G. Lefkowitz R.J. M. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar), M.G. Liggett S.B. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and other G-protein-coupled receptors, this region is considered important for receptor coupling to its G-protein, Indeed, the the Arg-389 and Gly-389 receptors is in functional coupling. binding of GTPγS to of coupling to Gs, was the two polymorphic with these accumulation of the high affinity in washed membrane in the absence of a guanine nucleotide was detected with the Arg-389 receptor could not be resolved in studies with the Gly-389 on the model T. P. D. Mol. Pharmacol. 1992; 41: Google Scholar, P. S. T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) of as well as the an basal of adenylyl cyclase to in the absence of also be with the Arg-389 receptor if has a of the in the presence of in was the The differences in basal activities that we were and the likely results from the low levels of expression utilized in the functional studies. we the functional phenotype as in A to be of signaling in cells that express the two polymorphic The in this region of the β1AR other are in The amino acid to position of the as well as the are in to with the deviation at position being found in the where Gly was we genetic variability in these other the high of in this its in G-protein and the and of the Gly to Arg are consistent with this variation functional β1AR are expressed on a number of cell types in the In the β1AR the βAR and is expressed on of the and where they to increase the and of in response to sympathetic It is to the of the β1AR polymorphisms at position in cardiac β1AR expression and in human heart M.R. M. P. Shah P. S. Res. PubMed Scopus Google Scholar, M.R. Mol. Pharmacol. Scholar). This response is to be a the heart from sympathetic in the of In of the of β1AR may to the the differences in the two β1AR polymorphisms that the of heart may be by the β1AR Indeed, is interindividual variation in the of heart as well as the cardiac response to used Am. J. PubMed Scopus Google Scholar, D. 1993; PubMed Scopus Google Scholar, Am. J. PubMed Scopus Google Scholar). βAR antagonists are utilized in the of heart the basis of is of the aforementioned of sympathetic interindividual variation in the response to in with heart J. Pharmacol. Google Scholar). on current be predicted that individuals the Arg-389 receptor be to they have a determined β1AR that a of adenylyl A be in the of with where could be predicted by β1AR These to be in human studies. we are of therapeutic the functional of the two β1ARs may be the basis of these polymorphisms as in we have to the frequencies of these β1AR polymorphisms in that polymorphisms are common in the is that represents the basis of These polymorphisms in such as heart and In we have found polymorphic variation of the human β1AR at amino acid 389, where Gly considered or Arg can be This variation receptor-Gs manifested as differences the two receptors in adenylyl cyclase activation. variation may a genetic basis for interindividual differences in or or the response to the β1AR.