Regulatory Properties of α_1B-Adrenergic Receptors Defective in Coupling to Phosphoinositide Hydrolysis

Abstract

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for ␣ 1B -adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and downregulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The ⌬12 and ⌬5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (⌬[246 -261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by ␣ 1B -adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.

Up-Regulation of α1B-Adrenergic Receptors with Defects in G Protein Coupling: Ligand-Induced Protection from Receptor Instability

“…Up-regulation occurred at quite low concentrations of epinephrine, the EC 50 value for epinephrine for inducing up-regulation in 24-h assays being 6 Ϯ 1 nM (Fig. 1B), consistent with the highaffinity binding of epinephrine to the ⌬12 receptors reported previously (Wang et al, 2002).…”

“…In contrast, exposure of cells expressing the wild-type ␣ 1B -AR to epinephrine in the same experiments consistently led to down-regulation, which was also caused by a decrease in B max with no change in affinity; the average decrease in B max was 43 Ϯ 11% (n ϭ 3), and the average K D values were 30 Ϯ 5 pM for control cells and 29 Ϯ 5 pM for epinephrine-pretreated down-regulated cells (data not shown). The lower affinity for [ 3 H]prazosin of the ⌬12 receptor compared with the wild-type receptor also has been reported previously (Wang et al, 2002).…”

“…The ⌬12 receptor contains a three-amino acid deletion, residues 227 to 229, YIV, at the N terminus of the third intracellular loop, whereas the ⌬5 receptor has 35 amino acids deleted, residues 227 to 261 (Wu et al, 1995). We focused on the ⌬12 receptor for further studies of up-regulation, because it contains a much smaller deletion than ⌬5 but exhibits an essentially identical phenotype in terms of G protein coupling, internalization, and up-regulation (Wang et al, 2002). The term up-regulation is used here to denote an increase in the radioligand binding capacity of the receptors, which may or may not be associated with an increase in the number of receptor protein molecules.…”

“…We recently investigated the sequestration and endocytosis properties of mutated ␣ 1B -ARs that are defective in G protein coupling and activation of the phosphoinositide (PI) hydrolysis pathway (Wang et al, 1997(Wang et al, , 2002. Three of these mutated ␣ 1B -ARs, Y348A, ⌬12, and ⌬5, were able to undergo agonist-induced sequestration within the plasma membrane but did not exhibit endocytosis into intracellular vesicles.…”

“…Intact Cell Incubations to Induce Up-Regulation. Up-regulation assays were performed as in our previous study (Wang et al, 2002). Cells plated on 100-mm dishes were treated on day 3 with 1 mM ascorbate for control cells or with epinephrine or other ligands in ascorbate to induce up-regulation.…”