Agonist-Stimulated and Tonic Internalization of Metabotropic Glutamate Receptor 1a in Human Embryonic Kidney 293 Cells: Agonist-Stimulated Endocytosis Is -Arrestin1 Isoform-Specific

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 -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 -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 -arrestin1 in HEK 293 cells. However, even though -arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither -arrestin1 nor -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, -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, 2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with -arrestin in response to agonist, the agonistindependent internalization of mGluR1a involves the -arrestinindependent targeting of mGluR1a to clathrin-coated vesicles. Metabotropic glutamate receptors (mGluRs) are members of the G protein-coupled receptor (GPCR) superfamily that are activated by the excitatory amino acid glutamate (Nakanishi, 1994). Glutamate is the major excitatory neurotransmitter in the central nervous system and is responsible for regulating integrative brain functions such as cognition, motor control, and neuronal development (Nakanishi, 1994). In addition to activating mGluRs, glutamate activates ionotropic glutamate receptors that are cation-specific ion channels (Nakanishi, 1994). Whereas ionotropic glutamate receptors mediate fast excitatory glutamate responses, mGluRs mediate slower glutamate responses by regulating the activity of intracellular second messenger cascades (Nakanishi, 1994). Consequently, mGluR activation is translated into long-lasting changes in synaptic activity (Aiba et al., 1994; Ichise et al., 2000). The mGluR family of GPCRs consists of eight receptor subtypes that are subclassified into three groups on the basis of sequence homology, pharmacology, and G protein coupling specificity (Nakanishi, 1994). Group 1 mGluRs (mGluR1 and mGluR5) are coupled via Gq to the stimulation of phospholipase C , leading to increases in intracellular inositol 1,4,5triphosphate formation, the release of calcium from intracellular stores, and the activation of protein kinase C (Nakanishi, 1994). Group 2 (mGluR2 and mGluR3) and group 3 (mGluR4, mGluR6, mGluR7 and mGluR8) are each negatively coupled to adenylyl cyclase (Nakanishi, 1994). S.S.G.F. is the recipient of a McDonald Scholarship Award from the Heart and Stroke Foundation of Canada. M.B. is the recipient of a CIHR fellowship. J.L.S. is the recipient of an Ontario Graduate Scholarship in Science and Technology. P.H.A. is the recipient of a Canadian Hypertension Society/Bristol-Myers Squibb/Sanofi/Canadian Institutes of Health Research (CIHR) fellowship. This work was supported by CIHR Grant MA-15506 to S.S.G.F. ABBREVIATIONS: mGluR, metabotropic glutamate receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; MEM, minimal essential medium; 4C3HPG, 4-carboxy-3-hydroxy-phenylglycine; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; HBSS, HEPES-buffered saline solution; IP, inositol phosphate; TBS, Tris-buffered saline; GFP, green fluorescent protein; 2AR, 2-adrenergic receptor. 0026-895X/01/6006-1243–1253$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 6 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 1061/948840 Mol Pharmacol 60:1243–1253, 2001 Printed in U.S.A. 1243 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from Members of the mGluR family bear no sequence or structural homology to other GPCRs (except calcium-sensing and GABAB receptors) other than the retention of a seven transmembrane topology characteristic of GPCRs. However, the molecular mechanisms contributing to the desensitization of “classical” GPCRs are conserved for this unique class of GPCR (Gereau and Heineman 1998; Dale et al., 2000; Sallese et al., 2000). In particular, group 1 mGluRs are phosphorylated by both second messenger-dependent protein kinases (Gereau and Heinemann, 1998) and G protein-coupled receptor kinases (GRKs) (Dale et al., 2000; Sallese et al., 2000). GPCR desensitization by both second messenger-dependent protein kinases and GRKs represents an important mechanism by which receptor activity is attenuated (Ferguson, 2001). GRK-mediated phosphorylation promotes the binding of -arrestin proteins that function to both uncouple GPCRs from their cognate heterotrimeric G proteins (Lohse et al., 1990; Attramadal et al., 1992) and target GPCRs to clathrin-coated pits for internalization (Ferguson et al., 1996; Zhang et al., 1996). The overexpression of either -arrestin1 or -arrestin2 has been demonstrated to augment the desensitization and internalization of several GPCRs (Pippig et al., 1993; Zhang et al., 1998; Oakley et al., 1999). Although both -arrestin isoforms interact with the 2-adaptin subunit of the AP2 adaptor complex to target GPCRs for endocytosis via clathrin-coated vesicles (Laporte et al., 2000), -arrestin2 has emerged as the principal GPCR endocytic adaptor protein (Oakley et al., 2000; Kohout et al., 2001). In addition, although -arrestins regulate the internalization of many GPCRs, there is evidence supporting the existence of a -arrestin-insensitive GPCR endocytic mechanism (reviewed by