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Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and gamma-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether beta-arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by beta-arrestin1 in HEK 293 cells. However, even though beta-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither beta-arrestin1 nor beta-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, beta-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, beta2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with beta-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the beta-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.

Previous studies have demonstrated that the interaction of the angiotensin II type 1A receptor (AT 1A R) carboxyl-terminal tail with Rab5a may modulate Rab5a activity, leading to the homotypic fusion of endocytic vesicles. Therefore, we have investigated whether AT 1A R/Rab5a interactions mediate the retention of AT 1A R⅐␤-arrestin complexes in early endosomes and whether the overexpression of Rab7 and Rab11 GTPases influences AT 1A R lysosomal degradation and plasma membrane recycling. We found that internalized AT 1A R was retained in Rab5a-positive early endosomes and was neither targeted to lysosomes nor recycled back to the cell surface, whereas a mutant defective in Rab5a binding, AT 1A R-(1-349), was targeted to lysosomes for degradation. However, the loss of Rab5a binding to the AT 1A R carboxyl-terminal tail did not promote AT 1A R recycling. Rather, it was the stable binding of ␤-arrestin to the AT 1A R that prevented, at least in part, AT 1A R recycling. The overexpression of wild-type Rab7 and Rab7-Q67L resulted in both increased AT 1A R degradation and AT 1A R targeting to lysosomes. The Rab7 expression-dependent transition of "putative" AT 1A R⅐␤-arrestin complexes to late endosomes was blocked by the expression of dominant-negative Rab5a-S34N. Rab11 overexpression established AT 1A R recycling and promoted the redistribution of AT 1A R⅐␤-arrestin complexes from early to recycling endosomes. Taken together, our data suggest that Rab5, Rab7, and Rab11 work in concert with one another to regulate the intracellular trafficking patterns of the AT 1A R.The angiotensin II type 1A receptor (AT 1A R) 1 is a member of the G protein-coupled receptor (GPCR) superfamily, the largest family of integral membrane receptor proteins. The AT 1A R is coupled via G q to the stimulation of phospholipase C␤, leading to increases in intracellular diacylglycerol and inositol 1,4,5-triphosphate and the release of calcium from intracellular stores (1). Agonist activation of the AT 1A R also leads to the desensitization of AT 1A R second messenger responses and the removal of cell-surface AT 1A R into the intracellular compartment of the cell (2-4). The agonist-stimulated desensitization and endocytosis of many GPCRs is initiated by GPCR kinasemediated phosphorylation, followed by ␤-arrestin binding (5). Both ␤-arrestin-dependent and ␤-arrestin-independent mechanisms of AT 1A