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To investigate the physiological role of the α1D-adrenergic receptor (α1D-AR) subtype, we created mice lacking the α1D-AR (α1D–/–) by gene targeting and characterized their cardiovascular function. In α1D–/– mice, the RT-PCR did not detect any transcript of the α1D-AR in any tissue examined, and there was no apparent upregulation of other α1-AR subtypes. Radioligand binding studies showed that α1-AR binding capacity in the aorta was lost, while that in the heart was unaltered in α1D–/– mice. Non-anesthetized α1D–/– mice maintained significantly lower basal systolic and mean arterial blood pressure conditions, relative to wild-type mice, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. Besides hypotension, the pressor responses to phenylephrine and norepinephrine were decreased by 30–40% in α1D–/– mice. Furthermore, the contractile response of the aorta and the pressor response of isolated perfused mesenteric arterial beds to α1-AR stimulation were markedly reduced in α1D–/– mice. We conclude that the α1D-AR participates directly in sympathetic regulation of systemic blood pressure by vasoconstriction

The difference in the expression of the alpha(1)-AR subtype with the patient may be the cause of the difference in the effectiveness of several subtype-selective alpha(1)-AR antagonists from patient to patient. The increase of alpha(1)-AR mRNA expression level with age could be an important factor in the pathogenesis of clinically significant BPH.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the ␣ 1B -adrenergic receptor (173 amino acids, amino acids 344 -516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369 -378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with fulllength ␣ 1B -adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the ␣ 1B -adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the ␣ 1B -adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that coexpressed ␣ 1B -adrenergic receptors and gC1q-R, most of the ␣ 1B -adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the ␣ 1B -adrenergic receptors.G protein-coupled receptors interact with several classes of cytoplasmic proteins including heterotrimeric G proteins, kinases, phosphatases, and arrestins, and the binding of cytoplasmic protein with the receptor regulates receptor signaling (1-4). These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5-8). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9) and with the Na ϩ /H ϩ -exchange regulatory factor (10). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor.We conducted a search for novel proteins that interact with the ␣ 1B -adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11)(12)(13)(14). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1 interacts with ␣ 1B -adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express ␣ 1B -adrenergic receptors and gC1q-R, the subcellular localization of ␣ 1B -adrenergic receptors is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as ...

To study the functional role of individual ␣ 1 -adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the ␣ 1B -AR and/or ␣ 1D -AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the ␣ 1D -AR knockout and ␣ 1B -/ ␣ 1D -AR double knockout mice, but not the ␣ 1B -AR knockout mice, had significantly (p Ͻ 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p Ͻ 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in ␣ 1B -/␣ 1D -AR double knockout mice. In an attempt to further examine ␣ 1 -AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, ␣ 1B -AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking ␣ 1D -AR had significantly (p Ͻ 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that ␣ 1B -and ␣ 1D -AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional ␣ 1D -AR, not ␣ 1B -AR, leads to an antihypertensive effect. The study shows differential contributions of ␣ 1B -and ␣ 1D -ARs in BP regulation.Catecholamines released from sympathetic nerve terminals cause vascular smooth muscle contraction primarily by activating ␣ 1 -adrenergic receptors (␣ 1 -ARs) in arteries (Hoffman, 2001). Thus, blockade of ␣ 1 -AR leads to a fall in peripheral vascular resistance. Because of their consistent effect in lowering systemic blood pressure (BP), ␣ 1 -AR blockers have been widely used as an antihypertensive drug. However, a large clinical trial unexpectedly disclosed that doxazosin, a nonselective ␣ 1 -AR antagonist, was associated with an increased incidence of heart failure (ALLHAT Collaborative Research Group, 2000). This raised a serious concern about the long-term use of ␣ 1 -AR antagonists in the treatment of hypertension (HT) (ALLHAT Collaborative Research Group, 2000). On the other hand, clinical efficacy of a subtypeselective inhibition of ␣ 1 -AR has not been fully determined.This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan, the Japan Health Science Foundation and Ministry of Human Health and Welfare.C.H. and T.K. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.007500. ABBREVIATIONS: ␣

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The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.

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