Kersten M. Small

A four amino acid deletion polymorphism in the third intracellular loop of the human alpha 2C-adrenergic receptor confers impaired coupling to multiple effectors.

The α2-adrenergic receptors (α2ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the α2AR subtype localized to human chromosome 4 (the pharmacologic α2CAR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322–325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human α2CAR and the polymorphic receptor. The Del322–325 variant had decreased high affinity agonist binding (KH = 7.3 ± 0.95 versus3.7 ± 0.43 nm; %RH = 31 ± 4 versus 49 ± 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to Gi, inhibiting adenylyl cyclase by 10 ± 4.3% compared with 73 ± 2.4% for wild-type α2CAR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase (∼71% impaired) and inositol phosphate production (∼60% impaired) was also found with the polymorphic receptor. The Del322–325 receptor was ∼10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322–325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.

Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization.

A polymorphic variant of the human α2B-adrenergic receptor (α2BAR), which consists of a deletion of three glutamic acids (residues 301–303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301–303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the α2BARs and GRK2 revealed that the Del301–303 receptor displayed ∼56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301–303 α2BAR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.

Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2–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.

Cardiac Specific Overexpression of Transglutaminase II (Gh) Results in a Unique Hypertrophy Phenotype Independent of Phospholipase C Activation

Tissue type transglutaminase (TGII, also known as Gh) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function, which is proposed as a coupling mechanism between α1-adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity in the highest expressing line was ∼37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [3H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to α1-adrenergic receptor stimulation; nor was protein kinase Cα (PKCα) or PKCε activity enhanced in the TGII transgenic mice. This is in contrast to mice moderately (∼5-fold) overexpressing Gαq, where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a remodeling of the heart with mild hypertrophy, elevated expression of β-myosin heavy chain and α-skeletal actin genes, and diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to β-agonist was not impaired. This set of pathophysiologic findings is distinct from that evoked by overexpression of Gαq. We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is not supported by these studies.

Identification and functional characterization of α2-adrenoceptor polymorphisms

For each alpha(2)-adrenoceptor subtype (alpha(2A), alpha(2B) and alpha(2C)), sequence variations within the coding region of each gene have been identified in humans. These result in substitutions or deletions of amino acids in the third intracellular loops of each receptor. This article summarizes the genetics and molecular biology of alpha(2)-adrenoceptor polymorphisms, including the consequences of each polymorphism on receptor signaling, as determined in transfected cells. These effects include alterations in G-protein coupling, desensitization and G-protein receptor kinase-mediated phosphorylation. Studies so far provide the mechanistic basis for future studies to investigate genetic risk factors and pharmacogenetics in pathophysiological conditions linked to alpha(2)-adrenoceptor function.

Identification of adrenergic receptor polymorphisms

Publisher Summary Adrenergic receptors (AR) are cell surface G protein-coupled receptors (GPCRs) that transduce signals due to binding of the endogenous catecholamines epinephrine and norepinepluine. Adrenergic receptors are the receptor component of the sympathetic nervous system, which regulates a host of homeostatic functions, including those of the cardiovascular, pulmonary, endocrine, and central nervous systems. Adrenergic receptors are also targets for a number of pharmacologic agents acting as agonists or antagonists. Genetic variation of GPCRs appears to be the basis for individual differences in physiologic responses or the efficacy of pharmacologic agents targeting this large class of proteins. This chapter presents detailed methods for the rapid detection of known polymorphisms of selected adrenergic receptors, which should facilitate additional physiologic and pharmacogenetic studies. Specific methods used to determine sequence variants in these receptors are discussed in the chapter. In addition, techniques for detecting specific polymorphisms and the consequences of these polymorphisms on signal transduction are briefly discussed in the chapter.

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

AbstractIntroduction: 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 eceptor (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 ≥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.

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 (∼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.

Genetic Variation Within the β1-Adrenergic Receptor Gene Results in Haplotype-Specific Expression Phenotypes

Cardiac β1-adrenergic receptor (β1AR) responsiveness in heart failure exhibits interindividual variation that may be attributable to polymorphisms of the intronless β1AR 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 β1AR polymorphisms, within the context of haplotypes, on receptor expression. Fifteen polymorphisms within the 6.1-kb gene with allele frequencies ≥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 β1AR 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 β1AR haplotypes, or haplotype expression clusters, as compared with individual polymorphisms.

Differential coupling of Arg- and Gly389 polymorphic forms of the β1-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.