Abstract
Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on alpha(1a)-adrenergic receptor (alpha(1a)AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human alpha(1a)AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the alpha(1a-1)AR isoform in human heart and prostate, we stably expressed an epitope-tagged alpha(1a-1)AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human alpha(1a)AR-mediated inositol phosphate signaling is acutely desensitized in response to both agonist and phorbol 12-myristate 13-acetate (PMA) exposure. Concurrent with desensitization, alpha(1a)ARs in (32)P(i)-labeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize alpha(1a)ARs when directly activated with PMA, inhibitors of PKC have no effect on agonist-mediated desensitization. In contrast, involvement of GRK kinases is suggested by the ability of GRK2 to desensitize alpha(1a)ARs. Internalization of cell surface alpha(1a)ARs also occurs in response to agonist stimulation (but not PKC activation), but is initiated more slowly than receptor desensitization. Significantly, deletion of the alpha(1a)AR carboxyl terminus has no effect on receptor internalization or either agonist-induced or GRK-mediated receptor desensitization. Because mechanisms underlying acute agonist-mediated regulation of human alpha(1a)ARs are primarily independent of the carboxyl terminus, they may be common to all functional alpha(1a)AR isoforms.
Highlights
Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on ␣1a-adrenergic receptor (␣1aAR) signaling and function
To address the issue of whether or not human ␣1aARs undergo acute desensitization in response to agonist stimulation, we examined the ability of HA-␣1a-expressing rat-1 fibroblasts to respond to a subsequent challenge with NE after initial pretreatment with NE
The intracellular carboxyl terminus has been implicated in regulation of several G protein-coupled receptors (GPCR) including the ␣1bAR, these data suggest that the carboxyl terminus of the human
Summary
Materials—Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were obtained from Invitrogen (Grand Island, NY). [125I]HEAT, [3H]inositol, [125I]iodoazidoprazosin, and [32P]orthophosphate were from PerkinElmer Life Sciences (Boston, MA). Measurement of Total Inositol Phosphate Production—Rat-1 cells stably expressing either HA-␣1a or HA-T348 receptors were labeled with [3H]inositol for 20 –24 h with 2.5 Ci/ml in DMEM supplemented with 3% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 g/ml). Following 18 h of transfection, cells were washed with Hank’s balanced salt solution, labeled with [3H]inositol in 10% fetal bovine serum, and assayed for total inositol phosphate production in DMEM essentially as described for stable cells. Different drugs were added during this incubation as indicated under “Results.” Following incubation, cells were washed three times with ice-cold PBS, solubilized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 mM Na3VO4, 10 mM Na4P2O7, 10 mM NaF, and Complete Protease Inhibitor Mixture (Roche Molecular Biochemicals)), and centrifuged at 14,000 ϫ g for 10 min at 4 °C. Statistical significance was analyzed by two-factor ANOVA (where appropriate) followed by a two-tailed unpaired t test, with p Ͻ 0.05 considered to be significant
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