Differential coupling of Arg- and Gly389 polymorphic forms of the beta1-adrenergic receptor leads to pathogenic cardiac gene regulatory programs.

The beta(1)-adrenergic receptor (beta(1)AR; ADRB1) polymorphism Arg389Gly is located in an intracellular loop and is associated with distinct human and mouse cardiovascular phenotypes. To test the hypothesis that beta(1)-Arg389 and beta(1)-Gly389 alleles could differentially couple to pathways beyond that of classic G(s)-adenylyl cyclase (AC)/cAMP signaling, we performed comparative gene expression profile analyses on hearts from wild-type and transgenic mice that expressed either human beta(1)-Arg389 or beta(1)-Gly389 receptors, or AC5, sampling at an early age prior to the onset of pathological features. All three models upregulated the expression of genes associated with RNA metabolism and translation and downregulated genes associated with mitochondria and energy metabolism, consistent with shared cAMP-driven increase in cardiac contractility, protein synthesis, and compensatory downregulation of mitochondrial energy production. Both beta(1)AR alleles activated additional genes associated with other pathways. Uniquely, beta(1)-Arg389 hearts exhibited upregulated expression of genes associated with inflammation, programmed cell death, and extracellular matrix. These observations expand the scope of 7-transmembrane domain receptor signaling propagation beyond known cognate G protein couplings. Moreover, they implicate alterations of a repertoire of processes evoked by a single amino acid variation in the cardiac beta(1)AR that might be exploited for genotype-specific heart failure diagnostics and therapeutics.

Genetic variation within the beta1-adrenergic receptor gene results in haplotype-specific expression phenotypes.

Cardiac beta1-adrenergic receptor (beta1AR) responsiveness in heart failure exhibits interindividual variation that may be attributable to polymorphisms of the intronless beta1AR gene. We sought to ascertain the polymorphisms of the full-length gene and the specific combinations of polymorphisms (haplotypes) in two reference populations. Using whole-gene transfections, we established the impact of beta1AR polymorphisms, within the context of haplotypes, on receptor expression. Fifteen polymorphisms within the 6.1-kb gene with allele frequencies > or =0.05 were found in the 5'-flanking and coding regions, but none in the 3'UTR. These were organized into six common haplotypes. Ethnic-specific and cosmopolitan polymorphisms and haplotypes were noted. Whole-gene transfections of A431 cells revealed an association between haplotype and expression, with as much as twofold differences in expression. Phenotypes clustered into three groups, representing high (two haplotypes), intermediate (three haplotypes), and low (one haplotype) expression. We conclude that the beta1AR gene is highly polymorphic and is commonly found in six haplotypic forms in the population. Receptor expression varies by haplotype, which provides the foundation for cardiovascular association studies with enhanced predictive power using beta1AR haplotypes, or haplotype expression clusters, as compared with individual polymorphisms.

Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2-adrenergic receptors within a unique heterodimeric complex.

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(2)AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP(1) receptor, since its endogenous agonist prostaglandin E(2) is abundant in the airway, but its functional implications are poorly defined. Activation of EP(1) failed to elicit ASM contraction in mouse trachea via this G(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), an effect accentuated by EP(1) agonist activation. Thus alone, EP(1) does not appear to have a significant direct effect on airway tone but acts as a modulator of the beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma.

Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment.

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.

Complex haplotypes derived from noncoding polymorphisms of the intronless alpha2A-adrenergic gene diversify receptor expression.

Alpha(2A)-adrenergic receptors (alpha(2A)AR) regulate multiple central nervous system, cardiovascular, and metabolic processes including neurotransmitter release, platelet aggregation, blood pressure, insulin secretion, and lipolysis. Complex diseases associated with alpha(2A)AR dysfunction display familial clustering, phenotypic heterogeneity, and interindividual variability in response to therapy targeted to alpha(2A)ARs, suggesting common, functional polymorphisms. In a multiethnic discovery cohort we identified 16 single-nucleotide polymorphisms (SNPs) in the alpha(2A)AR gene organized into 17 haplotypes of two major phylogenetic clades. In contrast to other adrenergic genes, variability of the alpha(2A)AR was primarily due to SNPs in the promoter, 5' UTR and 3' UTR, as opposed to the coding block. Marked ethnic variability in the frequency of SNPs and haplotypes was observed: one haplotype represented 70% of Caucasians, whereas Africans and Asians had a wide distribution of less common haplotypes, with the highest haplotype frequencies being 16% and 35%, respectively. Despite the compact nature of this intronless gene, local linkage disequilibrium between a number of SNPs was low and ethnic-dependent. Whole-gene transfections into BE(2)-C human neuronal cells using vectors containing the entire approximately 5.3-kb gene without exogenous promoters were used to ascertain the effects of haplotypes on alpha(2A)AR expression. Substantial differences (P < 0.001) in transcript and cell-surface protein expression, by as much as approximately 5-fold, was observed between haplotypes, including those with common frequencies. Thus, signaling by this virtually ubiquitous receptor is under major genetic influence, which may be the basis for highly divergent phenotypes in complex diseases such as systemic and pulmonary hypertension, heart failure, diabetes, and obesity.

Molecular properties and pharmacogenetics of a polymorphism of adenylyl cyclase type 9 in asthma: interaction between beta-agonist and corticosteroid pathways.

In asthma, the response to beta-agonists acting at beta2-adrenergic receptors (beta2AR) displays extensive interindividual variation. One effector for airway beta2AR, adenylyl cyclase type 9 (AC9), was considered a candidate locus for predicting beta-agonist efficacy in the absence and presence of corticosteroid treatment. One non-synonymous AC9 polymorphism has been identified, which results in substitution of Met for Ile at amino acid 772. Under standard culture conditions in stably transfected cells, we found decreased catalytic activity of Met772. However, cells cultured in the presence of glucocorticoid expressing Met772 had a significantly increased albuterol-stimulated adenylyl cyclase response (approximately 80%) when compared with those expressing Ile772 (approximately 20%, P=0.02). An equivalent increase in beta2AR expression was observed in both lines due to glucocorticoid, but AC9 expression was unaffected. The hypothesis that Met772-AC9 is associated with an improved albuterol bronchodilator response in asthmatics was investigated in 436 asthmatic children who were followed for 4 years and randomized to receive placebo or the inhaled corticosteroid budesonide. Met772 carriers on budesonide showed a significant improvement in forced expiratory volume in 1 s (P=0.005). Moreover, a highly significant interaction (P=0.002) was found for budesonide treatment and the AC9 polymorphism. These in vitro and human association studies are consistent with this AC9 polymorphism altering albuterol responsiveness in the context of concomitant inhaled corticosteroid administration, which is a common asthma regimen. The Met772-AC9 polymorphism represents one of most likely several multi-gene polymorphisms along the receptor-relaxation axis, which together may provide for a composite pharmacogenetic index for asthma therapy.

Polymorphisms of cardiac presynaptic alpha2C adrenergic receptors: Diverse intragenic variability with haplotype-specific functional effects.

The presynaptic alpha2C adrenergic receptors (AR) act to inhibit norepinephrine release in cardiac and other presynaptic nerves. We have recently shown that a genetic variant in the alpha2CAR coding region (Del322-325), which renders the receptor partially uncoupled from Gi, is a risk factor for heart failure. However, variability of heart failure phenotypes and a dominance of Del322-325 in those of African descent led us to hypothesize that other regions of this gene have functional polymorphisms. In a multiethnic population, we found 20 polymorphisms within 4,625 bp of contiguous sequence of this intronless gene encompassing the promoter, 5' UTR, coding, and 3' UTR. These polymorphisms occur in 24 distinct haplotypes with complex organizations, including multiple 5'-upstream polymorphisms in regions known to direct expression, a 3' UTR substitution polymorphism within an insertion/deletion sequence, and the radical coding polymorphism that deletes four amino acids. Relatively low linkage disequilibrium between many polymorphisms, few cosmopolitan haplotypes, prevalent ethnic-specific haplotypes, and substantial genetic divergence among haplotypes was noted. The dysfunctional Del322-325 allele was partitioned into multiple haplotypes, with frequencies of 48% to 2%. The functional implications of the haplotypes were ascertained by whole-gene transfections of human neuronal cells, where haplotype was significantly related (P < 0.001) to expression levels of receptor transcript and protein. Expression varied by as much as approximately 50% by haplotype, and such studies enabled haplotype clustering by phenotypic, rather than genotypic, similarities. Thus, depending on phenotype, expression-specific haplotypes may amplify, attenuate, or dominate the cardiomyopathic effect attributed to the alpha2CDel322-325 marker.

An Ile to Met polymorphism in the catalytic domain of adenylyl cyclase type 9 confers reduced beta2-adrenergic receptor stimulation.

Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled receptors such as the beta2-adrenergic receptor (beta2AR). Genetic variation in the receptor component of this pathway can alter signal transduction and the response to beta-agonists in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an Ile to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile772) and polymorphic (Met772) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met772 AC9 has lower basal and beta2AR-mediated adenylyl cyclase activities compared to Ile772 AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met772 membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta2AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma.

Gene and protein domain-specific patterns of genetic variability within the G-protein coupled receptor superfamily.

INTRODUCTION: Guanine nucleotide binding proteins (G-proteins) represent the targets for >50% of all therapeutics. There is substantial interindividual variation in response to agonists and antagonists directed to these receptors, which may, in part, be due to genetic polymorphisms. As a class, the sequence variability of G-protein-coupled receptor (GPCR) genes has not been characterized. STUDY DESIGN: This variability was investigated by sequencing promoter, 5'- and 3'-UTR, coding blocks, and intron-exon boundaries, of 64 GPCR genes in an ethnically diverse group of 82 individuals. RESULTS: Of the 675 single-nucleotide variations found, 61% occurred in > or =1% of the population sample and the nature of these 412 single nucleotide polymorphisms (SNPs) was assessed. 5'-UTR (p = 0.002) and coding (p = 0.006) SNPs were observed more often in GPCR genes, compared with 309 non-GPCR genes similarly interrogated. The prevalence of non-synonymous coding SNPs was unexpectedly high, with 65% of GPCR genes having at least one. Intron-containing genes had half as many non-synonymous coding SNPs compared with intronless genes (p = 0.0009), suggesting that when introns are not available coding regions provide sites for variation. A distinct relationship between the prevalence of non-synonymous SNPs and receptor structural domains was evident (p = 0.0006 by ANOVA), with variability being most prominent in the transmembrane spanning domains (38%) and the intracellular loops (24%). Phosphoregulatory domains, particularly the carboxy terminus, often the site for agonist-promoted phosphorylation by G-protein coupled receptor kinases, were the least polymorphic (8%). CONCLUSIONS: There is substantial genetic variability in potentially pharmacologically relevant coding and noncoding regions of GPCRs. Such variability should be considered in the development of new agents, or optimization of existing agents, targeted to these receptors.

Pharmacology and physiology of human adrenergic receptor polymorphisms.

Adrenergic receptors are expressed on virtually every cell type in the body and are the receptors for epinephrine and norepinephrine within the sympathetic nervous system. They serve critical roles in maintaining homeostasis in normal physiologic settings as well as pathologic states. These receptors are also targets for therapeutically administered agonists and antagonists. Recent studies have shown that at least seven adrenergic receptor subtypes display variation in amino acid sequence in the human population due to common genetic polymorphisms. Variations in potential regulatory domains in noncoding sequence are also present. Here, we review the consequences of these polymorphisms in terms of signaling, human physiology and disease, and response to therapy.

Synergistic polymorphisms of beta1- and alpha2C-adrenergic receptors and the risk of congestive heart failure.

BACKGROUND: Sustained cardiac adrenergic stimulation has been implicated in the development and progression of heart failure. Release of norepinephrine is controlled by negative feedback from presynaptic alpha2-adrenergic receptors, and the targets of the released norepinephrine on myocytes are beta1-adrenergic receptors. In transfected cells, a polymorphic alpha2C-adrenergic receptor (alpha2CDel322-325) has decreased function, and a variant of the beta1-adrenergic receptor (beta1Arg389) has increased function. We hypothesized that this combination of receptor variants, which results in increased synaptic norepinephrine release and enhanced receptor function at the myocyte, would predispose persons to heart failure. METHODS: Genotyping at these loci was performed in 159 patients with heart failure and 189 controls. Logistic-regression methods were used to determine the potential effect of each genotype and the interaction between them on the risk of heart failure. RESULTS: Among black subjects, the adjusted odds ratio for heart failure among persons who were homozygous for alpha2CDel322-325 as compared with those with the other alpha2C-adrenergic receptor genotypes was 5.65 (95 percent confidence interval, 2.67 to 11.95; P<0.001). There was no increase in risk with beta1Arg389 alone. However, there was a marked increase in the risk of heart failure among persons who were homozygous for both variants (adjusted odds ratio, 10.11; 95 percent confidence interval, 2.11 to 48.53; P=0.004). The patients with heart failure did not differ from the controls in the frequencies of nine short tandem-repeat alleles. Among white subjects, there were too few who were homozygous for both polymorphisms to allow an adequate assessment of risk. CONCLUSIONS: The alpha2CDel322-325 and beta1Arg389 receptors act synergistically to increase the risk of heart failure in blacks. Genotyping at these two loci may be a useful approach for identification of persons at risk for heart failure or its progression, who may be candidates for early preventive measures.

False positive non-synonymous polymorphisms of G-protein coupled receptor genes.

Polymorphisms of G-protein coupled receptor (GPCR) genes are associated with disease risk and modification, and the response to receptor-directed therapy. Genomic sequencing ( approximately 1700 automated runs) from as many as 120 chromosomes from 60 multiethnic individuals was performed to confirm non-synonymous coding polymorphisms reported in the dbSNP database from 25 randomly selected GPCR genes. These polymorphisms were in regions of the receptors responsible for structural integrity, ligand binding, G-protein coupling and phosphoregulation. However, most of these putative polymorphisms could not be confirmed (false positive rate of 68%). Based on these results, we suggest that the variability of the superfamily is not well defined, and we caution against exclusive reliance on databases for selection of candidate GPCR polymorphisms for disease association and pharmacogenetic studies.