Trafficking of ₂-adrenergic receptors: insulin and -agonists regulate internalization by distinct cytoskeletal pathways

Insulin and -adrenergic agonists stimulate a rapid phosphorylation and sequestration of the ₂-adrenergic receptors (₂ARs). Although the expectation was that a common pathway would be involved in the trafficking of the ₂AR in response to either hormone, studies reported herein show the existence of unique cytoskeletal requirements for internalization/recycling of G-protein-coupled receptors, such as the ₂AR. Treatment of human epidermoid carcinoma A431 cells with nocodazole, which binds tubulin monomer in vivo and catalyzes the depolymerization of microtubules, effectively blocks -adrenergic agonist-induced, but not insulin-induced, sequestration of ₂ARs. Treatment with latrunculin-A, an agent that sequesters actin monomer and leads to loss of actin filaments, had no effect on the ability of -adrenergic agonists to stimulate internalization of ₂ARs, but blocked the ability of insulin to stimulate counterregulation of ₂ARs via internalization. Although nocodazole had no effect on insulin-stimulated sequestration of ₂ARs, the recycling of the internalized receptors to the cell membrane was sensitive to depolymerization of microtubules by this agent. Latrunculin-A, by contrast, blocks the recycling of ₂ARs internalized in response to -agonist, while attenuating recycling of receptors internalized in response to insulin stimulation. These data show the existence of unique cytoskeletal requirements for G-protein-coupled-receptor trafficking in response to agonist compared with a counterregulatory hormone, and for sequestration versus recycling of the receptors to the cell membrane.