Novel human alpha1a-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function.
ABSTRACT: We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the alpha(1a)-adrenoceptor (alpha(1a)AR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type alpha(1a)AR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose-response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for alpha(1a)AR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor-G protein coupling. In conclusion, four naturally-occurring human alpha(1a)AR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide alpha(1a)AR SNP choice for future clinical studies.
Project description:We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.
Project description:We compared the role of tyrosine kinases in alpha(1A)-adrenergic receptor (AR) and growth factor receptor stimulation of mitogen-activated protein kinase pathways in PC12 cells. Norepinephrine (NE) (noradrenaline), epidermal growth factor (EGF) and nerve growth factor (NGF) caused different patterns of tyrosine phosphorylation in PC12 cells stably expressing alpha(1A)-ARs. NE increased tyrosine phosphorylation of focal adhesion-related kinase Pyk2 and a 70 kDa protein, probably paxillin, whereas EGF strongly stimulated tyrosine phosphorylation of the EGF receptor and cytokine-activated kinase Jak2. The EGF receptor inhibitor AG1478 inhibited activation of extracellular signal-regulated kinases (ERKs) by EGF but not by NE. EGF and NGF strongly activated tyrosine phosphorylation of Shc and caused association of Src-homology collagen (Shc) with growth-factor-receptor-bound protein 2 (Grb2); however, neither NE nor UTP caused substantial activation of the Shc/Grb2 pathway. NE, UTP, EGF and NGF all increased tyrosine phosphorylation of Src, and this was inhibited by the Src inhibitor PP2. However, PP2 inhibited ERK activation in response to NE and UTP, but not in response to EGF or NGF. PP2 also completely blocked NE-induced PC12 cell differentiation, but had no measurable effect on NGF-induced differentiation. These studies show that activation of mitogen-activated protein kinase pathways by G-protein-coupled receptors and tyrosine kinase receptors proceed through distinct molecular pathways in PC12 cells, and support an obligatory role for Src activation in mitogenic responses to alpha(1A)-ARs in these cells.
Project description:BACKGROUND: Agonistic autoantibodies to the alpha(1)-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. METHODS/PRINCIPAL FINDINGS: We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A)-adrenergic receptor and maintained them for one year. Alpha(1A)-adrenergic antibodies (alpha(1A)-AR-AB) were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A)-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max) demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min). Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang) II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. CONCLUSIONS/SIGNIFICANCE: We show that alpha(1A)-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A)-AR-AB could contribute to cardiovascular endorgan damage.
Project description:We previously identified a naturally occurring human SNP, G247R, in the third intracellular loop of the ?(1a)-adrenergic receptor (?(1a)-247R) and demonstrated that constitutive expression of ?(1a)-247R results in twofold increased cell proliferation compared with WT. In the present study we elucidate molecular mechanisms and signal transduction pathways responsible for increased cell proliferation unique to ?(1a)-247R, but not ?(1a)-WT, ?(1b), or ?(1d)AR subtypes. We show that elevated levels of matrix metalloproteinase-7 (MMP7) and a disintegrin and metalloproteinase-12 (ADAM12) in ?(1a)-247R-expressing cells are responsible for EGF receptor (EGFR) transactivation, downstream ERK activation, and increased cell proliferation; this pathway is confirmed using MMP, EGFR, and ERK inhibitors. We demonstrate that EGFR transactivation and downstream ERK activation depends on increased shedding of heparin-binding EGF. Finally, we demonstrate that knockdown of MMP7 or ?-arrestin1 by shRNAs results in attenuation of proliferation of cells expressing ?(1a)-247R. Importantly, accelerated cell proliferation triggered by the ?(1a)-247R is serum- and agonist-independent, providing unique evidence for constitutive active coupling to the ?-arrestin1/MMP/EGFR transactivation pathway by any G protein-coupled receptor. These findings raise the possibility of a previously unexplored mechanism for sympathetically mediated human hypertension triggered by a naturally occurring human genetic variant.
Project description:We have provided the first evidence for specific heteromerization between the ?(1A)-adrenoceptor (?(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. ?(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent ?-arrestin recruitment that was in turn dependent upon co-expression of ?(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent ?-arrestin recruitment was inhibited not only by the ?(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective ?-adrenoceptor antagonist with ?(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for ?-arrestin recruitment at the ?(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that ?(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.
Project description:Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.
Project description:RATIONALE:Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors. OBJECTIVE:Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist. METHODS AND RESULTS:In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the ?1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor. CONCLUSIONS:Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.
Project description:The ?1A-AR is thought to couple predominantly to the G?q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with G?q coupling-defective variants of ?1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between ?1A-AR and ?2-AR that leads to potentiation of a G?q-independent signaling cascade in response to ?1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as ?-AR-selective agonist, was examined with respect to activation of ?1A-AR. ?1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at ?1A-AR. Iso induced signaling at ?1A-AR was further interrogated by probing steps along the G?q /PLC, G?s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with ?1A-AR, and CHO_?1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by ?1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical G?q- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of ?1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to G?q.
Project description:Gene expression profiles in hearts of Lewis rats immunized with a peptide of human alpha1A-adrenergic receptor. The pathophysiological relevance of chronic autoantibodies against alpha-1A-adrenergic receptor stimulation in rats was investigated. Lewis rats were immunized using synthesized peptides of second extracellular loop of the human alpha-1A-adrenergic receptor and raised for one year. The gene expression in hearts of three immunized and three control rats were analyzed using Affymetrix Rat Genome 230 2.0 Arrays.
Project description:Arthralgias and myalgias are major side effects associated with aromatase inhibitor (AI) therapy of breast cancer. In a recent genome-wide association study, we identified SNPs - including one that created an estrogen response element near the 3' end of the T-cell leukemia 1A (TCL1A) gene - that were associated with musculoskeletal pain in women on adjuvant AI therapy for breast cancer. We also showed estrogen-dependent, SNP-modulated variation in TCL1A expression and, in preliminary experiments, showed that TCL1A could induce IL-17RA expression. In the present study, we set out to determine whether these SNPs might influence cytokine expression and effect more widely, and, if so, to explore the mechanism of TCL1A-related AI-induced side effects.The functional genomic experiments performed included determinations of TCL1A, cytokine and cytokine receptor expression in response to estrogen treatment of U2OS cells and lymphoblastoid cell lines that had been stably transfected with estrogen receptor alpha. Changes in mRNA and protein expression after gene knockdown and overexpression were also determined, as was NF-?B transcriptional activity.Estradiol (E2) increased TCL1A expression and, in a TCL1A SNP-dependent fashion, also altered the expression of IL-17, IL-17RA, IL-12, IL-12RB2 and IL-1R2. TCL1A expression was higher in E2-treated lymphoblastoid cell lines with variant SNP genotypes, and induction of the expression of cytokine and cytokine receptor genes was mediated by TCL1A. Finally, estrogen receptor alpha blockade with ICI-182,780 in the presence of E2 resulted in greatly increased NF-?B transcriptional activity, but only in cells that carried variant SNP genotypes. These results linked variant TCL1A SNP sequences that are associated with AI-dependent musculoskeletal pain with increased E2-dependent TCL1A expression and with downstream alterations in cytokine and cytokine receptor expression as well as NF-?B transcriptional activity.SNPs near the 3' terminus of TCL1A were associated with AI-dependent musculoskeletal pain. E2 induced SNP-dependent TCL1A expression, which in turn altered IL-17, IL-17RA, IL-12, IL-12RB2, and IL-1R2 expression as well as NF-?B transcriptional activity. These results provide a pharmacogenomic explanation for a clinically important adverse drug reaction as well as insights into a novel estrogen-dependent mechanism for the modulation of cytokine and cytokine receptor expression.