Constitutive internalization of G protein-coupled receptors and G proteins via clathrin-independent endocytosis.

Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the beta2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Galpha(s) and Galpha(q), localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Galpha(s) and Galpha(q) remained associated with the PM and these endosomal membranes, whereas beta2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.