Up-Regulation of α_1B-Adrenergic Receptors with Defects in G Protein Coupling: Ligand-Induced Protection from Receptor Instability

Abstract

The biochemical basis for the unexpected agonist-induced upregulation of the number of radioligand binding sites for two mutated ␣ 1B -adrenergic receptors reported previously was investigated. Up-regulation was independent of the expression vector used and was not prevented by cycloheximide or actinomycin D, eliminating several potential transcriptional mechanisms and new receptor protein synthesis. Antagonists were also able to induce up-regulation, suggesting that ligand occupancy without signal generation was sufficient to induce the increase in binding sites. Accordingly, we hypothesized that up-regulation results from ligand-induced protection from inherent instability of these mutated receptors. Studies with receptors in isolated membranes revealed that the two mutated receptors that exhibited up-regulation in intact cells also exhibited an inherent instability of their ligand binding capacity, and binding of either agonists or antagonists to these receptors could protect against the loss of binding. In contrast, the wildtype receptor and other mutated receptors that did not exhibit up-regulation in intact cells did not exhibit instability or ligandinduced protection in isolated membranes. The occurrence of instability and protection in isolated membranes for only those mutated receptors and ligands that exhibit up-regulation in intact cells provides compelling evidence that the apparent up-regulation of binding sites in intact cells results from ligandinduced protection from an inherent instability of these G protein coupling-defective receptors. Inclusion of protease inhibitors markedly reduced the loss of binding in isolated membranes, implicating membrane-localized proteolysis as the likely mechanism for the instability.

Addition of a signal peptide sequence to the α1D‐adrenoceptor gene increases the density of receptors, as determined by [3H]‐prazosin binding in the membranes

“…Whether the same mechanism of action underlies all of these four increases in the α 1D ‐AR populations is uncertain. A new concept is that GPCRs may have inherent instability and therefore become downregulated, and that antagonists may stabilize receptor conformations that are less prone to downregulation (Prinster et al ., 2003; Zeng et al ., 2003). Thus, not only blocking of constitutively active receptors but also stabilization of unstable receptors, could explain the previously observed antagonist‐mediated increment in membrane‐localized α 1D ‐adrenoceptors (McCune et al ., 2000; see Garcia‐Sainz & Villalobos‐Molina, 2004).…”

Adrenoceptor Desensitization: Current Understanding of Mechanisms

“…This indicated that phosphorylation by GRK is only a requirement for internalization. In contrast, mutations in the PKC phosphorylation sites in the C tail gave rise to "PKC-site mutants", which demonstrated partial impairment in sequestration and endocytosis but completely diminished down-regulation, indicating an important role for PKC in this process (Prinster, 2003).…”