Ca²⁺/Calmodulin‐Mediated Regulation of Agonist‐Induced Sequestration of G_q Protein‐Coupled Histamine H₁ Receptors in Human U373 MG Astrocytoma Cells

Abstract

Abstract: We investigated the regulation by intracellular Ca 2+ of agonist‐induced sequestration of G q protein‐coupled histamine H 1 receptors in human U373 MG astrocytoma cells. Histamine‐induced sequestration of H 1 receptors from the cell surface membrane was detected as the loss of [ 3 H]mepyramine binding sites on intact cells accessible to the hydrophilic H 1 ‐receptor antagonist pirdonium. The changes in the pirdonium‐sensitive binding of [ 3 H]mepyramine were mirrored by changes in the subcellular distribution of H 1 receptors detected by sucrose density gradient centrifugation. The histamine‐induced sequestration of H 1 receptors did not occur in hypertonic medium, in which clathrin‐mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca 2+ or in the presence of the calmodulin antagonists W‐7 and calmidazolium. Inhibitors of protein kinase C (H‐7 and GF109203X), Ca 2+ /calmodulin‐dependent protein kinase II (KN‐62), or protein phosphatase 2B (FK506) did not alter the time course of H 1 ‐receptor sequestration. These results provide the first evidence that agonist‐induced, clathrin‐mediated sequestration of G q protein‐coupled receptors is transiently inhibited by Ca 2+ /calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.