Phosphorylation Sites on Two Domains of the β2-Adrenergic Receptor Are Involved in Distinct Pathways of Receptor Desensitization

TLDR

Continuous exposure to agonists causes rapid desensitization, with low isoproterenol reducing sensitivity but not maximal response, while high concentrations diminish both sensitivity and maximal responsiveness in CHW cells expressing β2‑adrenergic receptors. The study aims to investigate how phosphorylation of β2AR at protein kinase A and β2AR kinase sites mediates desensitization at low versus high agonist concentrations. Researchers expressed three mutant β2AR constructs in CHW cells that lack putative phosphorylation sites for PKA and/or β2AR kinase to dissect the phosphorylation‑dependent desensitization pathways. Low agonist concentrations preferentially phosphorylate PKA sites, reducing sensitivity, whereas high concentrations induce phosphorylation at both PKA and β2AR kinase sites, leading to loss of maximal responsiveness, indicating distinct domain phosphorylation drives different desensitization mechanisms.

Abstract

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.