Acute Agonist-mediated Desensitization of the Human α1a-Adrenergic Receptor Is Primarily Independent of Carboxyl Terminus Regulation

Abstract

Despite important roles in myocardial hypertrophy and benign prostatic hyperplasia, little is known about acute effects of agonist stimulation on ␣ 1a -adrenergic receptor (␣ 1a AR) signaling and function. Regulatory mechanisms are likely complex since 12 distinct human ␣ 1a AR carboxyl-terminal splice variants have been isolated. After determining the predominance of the ␣ 1a-1 AR isoform in human heart and prostate, we stably expressed an epitope-tagged ␣ 1a-1 AR cDNA in rat-1 fibroblasts and subsequently examined regulation of signaling, phosphorylation, and internalization of the receptor. Human ␣ 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, ␣ 1a ARs in 32 P ilabeled cells are rapidly phosphorylated in response to both NE and PMA stimulation. Despite the ability of PKC to desensitize ␣ 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 ␣ 1a ARs. Internalization of cell surface ␣ 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 ␣ 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 ␣ 1a ARs are primarily independent of the carboxyl terminus, they may be common to all functional ␣ 1a AR isoforms.␣ 1a -Adrenergic receptors (␣ 1a ARs) 1 are G protein-coupled receptors (GPCR) that mediate sympathetic nervous system responses such as smooth muscle contraction and myocardial inotropy (1). NE stimulation of ␣ 1 ARs predominantly activates G q and results in membrane polyphosphoinositide hydrolysis by activation of phospholipase C␤; the resultant second messengers IP 3 and DAG mobilize intracellular calcium and activate protein kinase C (PKC), respectively (2). Three ␣ 1 AR subtypes (␣ 1a , ␣ 1b , and ␣ 1d ) have been cloned and pharmacologically characterized in several expression systems (for review, see Ref.2). Clinically, activation of ␣ 1a ARs has been implicated in the dynamic component of benign prostatic hyperplasia leading to bladder outlet obstruction and in the development of myocardial hypertrophy (3-5). Notwithstanding the importance of ␣ 1a ARs in several pathophysiological states, surprising little is known about mechanisms underlying ␣ 1a AR expression and function. Transcriptional mechanisms unique to ␣ 1a ARs have been shown to be important in maintaining full ␣ 1 AR responsiveness to agonist in rat neonatal myocytes where long term (24 -72 h) NE stimulation leads to up-regulation ␣ 1a AR mRNA and receptor protein expression, concurrent with down-regulation of ␣ 1b and ␣ 1d AR subtypes (5, 6). While important, such long-term studies do not examine...

Mammalian Tolloid Alters Subcellular Localization, Internalization, and Signaling of α1a-Adrenergic Receptors

“…The number of cell surface receptors initially increased during the first 5 min of agonist stimulation and then decreased by 58% by 60 min. Therefore, the results were consistent with the internalization pattern of ␣ 1A -AR in rat-1 fibroblast cells (Price et al, 2002). Price et al (2002) observed that although the number of cell-surface wild-type ␣ 1 -AR showed little increase in the early stages of agonist stimulation, the number of carboxyl-terminally truncated mutant ␣ 1 -AR T348 increased significantly during the same period.…”

“…Therefore, the results were consistent with the internalization pattern of ␣ 1A -AR in rat-1 fibroblast cells (Price et al, 2002). Price et al (2002) observed that although the number of cell-surface wild-type ␣ 1 -AR showed little increase in the early stages of agonist stimulation, the number of carboxyl-terminally truncated mutant ␣ 1 -AR T348 increased significantly during the same period. Given that fibroblasts express mTLD naturally, whereas there was no mTLD expression at the mRNA level in HEK293 cells (data not shown), and considering our results that amino acid residues 322 to 359 are the primary binding region to CUB5 domain of mTLD, conflicting results of ␣ 1A -AR internalization from different groups are expected.…”

The α1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits

“…4 The time frame of receptor exit from the light rafts is much slower than the acute signaling response of the ␣ 1a AR. Indeed, evidence suggests G q -coupled receptors induce PIP 2 cleavage (47) and calcium release (56,57) within seconds, whereas maximal rates of IP signaling from activated ␣ 1a ARs conclude in less than 1 min (38). Similar short time frames are observed for agonist-mediated ␣ 1a AR desensitization and phosphorylation (38,57).…”

“…Cell Culture-Clonal cell lines of rat-1 fibroblasts expressing HA-␣ 1a AR (ϳ1 pmol/mg) have been described (37) as have rat-1 fibroblasts expressing HA-␣ 1a AR-EGFP (ϳ1 pmol/mg) (38). Cells were maintained in medium (10% heat-inactivated fetal bovine serum, penicillin/streptomycin, 0.5 mg/ml G418, and DMEM) at 37°C under 5% CO 2 .…”

COMBINED USE OF Α-Adrenergic AND MUSCARINIC ANTAGONISTS FOR THE TREATMENT OF VOIDING DYSFUNCTION

“…11 Although the carboxyl terminus of the α 1b -AR mediates the decreased expression, phosphorylation, internalization and desensitization of the receptor, the carboxyl terminus of α 1a -AR does not. 12 When expressed in CHO-K1 cells, all 4 functional splice variants as well as the WT of the α 1a -AR display the pharmacological properties of so-called α 1L -AR. 13 This finding proves that the distinct pharmacology of α 1L -AR in human LUT is not a function of the differential tissue expression of α 1a -AR splice variants and it strongly supports the concept that the distinct α 1L pharmacology of the human LUT is not the result of tissue expression of a different α 1 -AR, but rather a functional conformation of α 1a -AR.…”