Catecholamines stimulate cardiac contractility through beta(1)-adrenergic receptors (beta(1)-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased beta-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and G alpha(s) expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to beta-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to beta-receptor blockade.
The signaling impact of a human beta1-adrenergic receptor (beta1 AR) polymorphism at residue 49 of the aminoterminus (Ser-to-Gly substitution) was studied by recombinantly expressing each receptor. The two receptors displayed identical agonist and antagonist binding affinities. Furthermore, basal and agonist-stimulated adenylyl cyclase activities were the same for these receptors as assessed in both cell types. Although short-term agonist exposure resulted in similar degrees of receptor internalization, long-term agonist-promoted downregulation was greater for Gly49 compared with Ser49. The Gly49 receptor underwent a 24 +/- 3% loss of receptor density after exposure to isoproterenol for 18 h, whereas Ser49 underwent no such loss. In studies in which receptor synthesis was inhibited, agonist-promoted downregulation for Gly49 was 55 +/- 3% compared with 36 +/- 5% for Ser49. In the absence of agonist, degradative turnover of each receptor was the same. Immunoblotting revealed that some of the Ser49 receptor exists as a highly N-glycosylated form (approximately 105-kD molecular mass), which is not present with Gly49. Thus the phenotype of the Gly49 polymorphic receptor is that of wild-type coupling with enhanced agonist-promoted downregulation, which is associated with altered N-glycosylation. Based on this cellular phenotype, the beta1 AR Gly49 polymorphism may impart a beneficial effect in chronic heart failure.
Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human  1 -adrenergic receptor ( 1 AR) or a gain-of-function polymorphic variant ( 1 AR-Arg 389 ), and the  2 -adrenergic receptor ( 2 AR) or a loss-of-function polymorphic receptor ( 2 AR-Ile 164 ). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the  1 AR, the enhanced function of the Arg 389 receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized  1 AR-Arg 389 signaling was equivalent to non-desensitized wild-type  1 AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type  2 AR underwent less desensitization compared with  2 AR-Ile 164 , thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.Like a number of other G-protein-coupled receptors, the  1 -and  2 -adrenergic receptors ( 1 AR and  2 AR, 1 respectively) undergo desensitization during continuous exposure to agonist.Such desensitization occurs maximally after several minutes of agonist exposure and is due to decreased interaction with G s , which is evoked by receptor phosphorylation (1). Thus the signal transduction of these receptors can be characterized as one of two potential conditions or states, defined here as "control" (no recent exposure to agonist) and "desensitized." However, we have recently shown that an alteration in receptor-G s coupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the  1 AR gene in the human population (2, 3) results in either Gly or Arg being encoded at amino acid position 389 of the proximal portion of the cytoplasmic tail. In studies using transfected cells with equivalent expression of the two receptors, the  1 AR-Arg 389 displays an increase in G s coupling compared with  1 AR-Gly 389 (2). So, one can consider that the human  1 AR can exist in four agoniststimulated states: Gly 389 control and desensitized, and Arg 389 control and desensitized. And, since basal (non-ag...
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