The presynaptic ␣2C adrenergic receptors (AR) act to inhibit norepinephrine release in cardiac and other presynaptic nerves. We have recently shown that a genetic variant in the ␣2CAR coding region (Del322-325), which renders the receptor partially uncoupled from G i, 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 insertion͞ 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 Ϸ50% 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 ␣2CDel322-325 marker.T he adrenergic receptors (ARs), whose endogenous ligands are epinephrine and norepinephrine, are members of the superfamily of seven transmembrane spanning G proteincoupled receptors. ARs modulate a host of functions relative to the sympathetic nervous system, including neurotransmitter release, and cardiac, vascular, pulmonary, renal, metabolic, and central nervous system function. For over a decade, it has been known that the expression and͞or function of ARs varies considerably between individuals (1), even when studies are carried out under stringent conditions in normal individuals. Similarly, the physiologic or clinical response to receptor agonists and antagonists shows marked interindividual variability (2, 3). One explanation for such variability is that ARs are dynamically regulated by multiple mechanisms so as to maintain homeostasis, and that individuals may be under different environmental influences such that receptor expression or function is altered. However, considerable evidence has accumulated over the last few years suggesting that interindividual differences in receptor expression or function is based on genetic variability of the genes encoding these receptors (4).Of recent interest has been genetic variabil...

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.

␣2A-adrenergic receptors (␣2AAR) regulate multiple central nervous system, cardiovascular, and metabolic processes including neurotransmitter release, platelet aggregation, blood pressure, insulin secretion, and lipolysis. Complex diseases associated with ␣2AAR dysfunction display familial clustering, phenotypic heterogeneity, and interindividual variability in response to therapy targeted to ␣2AARs, suggesting common, functional polymorphisms. In a multiethnic discovery cohort we identified 16 single-nucleotide polymorphisms (SNPs) in the ␣2AAR gene organized into 17 haplotypes of two major phylogenetic clades. In contrast to other adrenergic genes, variability of the ␣2AAR 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 Ϸ5.3-kb gene without exogenous promoters were used to ascertain the effects of haplotypes on ␣2AAR expression. Substantial differences (P < 0.001) in transcript and cell-surface protein expression, by as much as Ϸ5-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.adenylyl cyclase ͉ G protein-coupled receptor ͉ pharmacogenetics ͉ sympathetic nervous system

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.