Cross Talk Between m3-Muscarinic and β₂-Adrenergic Receptors at the Level of Receptor Phosphorylation and Desensitization

Abstract

In this study we investigated cross talk between m3-muscarinic and β₂-adrenergic receptors coexpressed in Chinese hamster ovary (CHO-m3/β₂) cells, focusing on two possible mechanisms of regulation. The first mechanism is based on recent in vitro studies demonstrating that G protein-coupled receptor kinase (GRK) activity, the protein kinase responsible for β₂-adrenergic receptor homologous phosphorylation and desensitization, may be regulated by calcium/calmodulin and membrane phosphatidylinositol 4,5-bisphosphate. Stimulation of the phospholipase C signaling pathway via m3-muscarinic receptors in CHO-m3/β₂ cells increased intracellular free calcium by ∼10 fold and membrane phosphatidylinositol 4,5-bisphosphate levels decreased by ∼74%. However, despite these changes the ability of endogenous kinases, possibly the GRKs, to phosphorylate the β₂-adrenergic receptor was not altered. The second mechanism investigated involves a direct heterologous phosphorylation of the β₂-adrenergic receptor after muscarinic receptor stimulation. Activation of m3-muscarinic receptors did mediate heterologous phosphorylation of β₂-adrenergic receptors in a GRK-independent fashion, via protein kinase C. Heterologous β₂-adrenergic receptor phosphorylation correlated with receptor desensitization as measured by a loss in guanine-nucleotide sensitive-high affinity agonist binding and reduction in maximal cAMP response. This receptor cross talk may have a profound physiological importance in a wide variety of cell types, for example smooth muscle, where these two receptors are known to be coexpressed.