Signaling properties of human α1D-adrenoceptors lacking the carboxyl terminus: intrinsic activity, agonist-mediated activation, and desensitization

Abstract

alpha(1)-Adrenoceptors are differentially regulated by protein kinase C-mediated phosphorylation. The most sensitive member of this family is the alpha(1D)-subtype, which is also characterized by a constitutive activity and a reduced expression at the plasma membrane controlled by the amino terminus. Information on the structural domains that determine the function and regulation of this receptor subtype is scarce. Therefore, the function and phosphorylation of amino terminus-truncated (Delta1-79, (DeltaN)) alpha(1D)-adrenoceptors were studied and compared with those of alpha(1D)-adrenoceptors truncated both at the amino and carboxyl termini (Delta1-79 and Delta441-572, (DeltaN-DeltaC)). These receptors were stably expressed in rat-1 fibroblast, at relatively high density ( approximately 2 pmol/mg of membrane protein), and showed intrinsic activity that was markedly increased by noradrenaline. Interestingly, activation of protein kinase C markedly attenuated (desensitized) the function of both DeltaN and DeltaN-DeltaC alpha(1D)-adrenoceptors. These receptors were photolabeled and immunoprecitated with an antibody directed against an influenza hemagglutinin epitope inserted at the amino termini. Metabolic labeling with radioactive phosphate and receptor immunoprecipitation studies indicated that these receptors are phosphoproteins whose phosphorylation state is increased by noradrenaline and by activation of protein kinase C. Our data indicate that carboxyl terminus-truncated alpha(1D)-adrenoceptors are fully functional and subjected to regulation by phosphorylation. The roles of the carboxyl termini differ among alpha(1)-adrenoceptor subtypes.

Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway

“…This is in contrast with what has been observed with the α 1D -AR subtype, i. e., α 1D -AR-mediated calcium/phosphoinositide signaling, desensitization, and phosphorylation can take place in a carboxyl tail-truncated mutant (Rodríguez-Pérez et al, 2009). However, other pathways modulated by α 1D -ARs have not been studied.…”

“…In order to determine the role of the α 1D -adrenergic carboxyl terminus in signaling, a mutant with this deletion of the amino terminus (Δ1–79) (ΔN) and another with an additional carboxyl terminus deletion (Δ 441–572) (ΔNΔC) were expressed in Rat-1 cells (Rodríguez-Pérez et al, 2009) and changes in ERK phosphorylation and intracellular calcium were assessed. In cells expressing the ΔN mutant, we observed a subtle basal level of ERK phosphorylation that was markedly increased after 1 and 5 min of noradrenaline stimulation, and returned to near baseline after 30 min (Fig.…”

“…Plasmids coding for HA-tagged human α 1D -adrenoceptor (Δ1–79, amino terminus-truncated, ΔN) and (Δ1–79 and 441–572, amino and carboxyl termini-truncated, ΔNΔC) were previously reported (Rodríguez-Pérez et al, 2009). Plasmid coding for carboxyl terminus GFP-tagged proteins of the mutants described above (ΔN-GFP and ΔNΔC-GFP) were synthesized commercially by Mutagenex Inc.…”

“…Generation of Rat-1 cell lines expressing the α 1D -adrenoceptor deletion mutants was previously reported (receptor density ~ 1500 fmol/mg membrane protein) (Rodríguez-Pérez et al, 2009). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, G418 300 µg/ml, streptomycin 100 µg/ml, penicillin 100 U/ml and amphotericin B 0.25 µg/ml.…”

“…Intracellular calcium concentration was assessed as previously described (Rodríguez-Pérez et al, 2009). In brief, cells were serum-starved overnight, then loaded with 2.5 µM of the fluorescent Ca 2+ indicator, Fura-2/AM, in Krebs-Ringer-HEPES containing 0.05% bovine serum albumin, pH 7.4 for 1 h at 37°C, and then washed three times to eliminate unincorporated dye.…”