Agonist‐induced internalization of the metabotropic glutamate receptor 1a is arrestin‐ and dynamin‐dependent

Abstract

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) traf®cking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 mM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quanti®cation of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin-and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319±418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 mM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-22green¯uorescent protein (arrestin-22GFP) or arrestin-32GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin-and dynamin-dependent process. Keywords: arrestin, dynamin, internalization, metabotropic glutamate receptor. Widely distributed throughout the CNS, metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that play a critical role in glutamate-mediated neurotransmission and synaptic plasticity events (for review, see Schoepp et al. 1999). On the basis of their amino acid sequence similarity, pharmacology and signal transduction mechanisms, mGluRs have been classi®ed into three groups, with the group I mGluRs, mGluR1 and mGluR5, being coupled to G q and phospholipase C. Five splice variants of mGluR1 have been described, all of them differing in the length of their C-terminal tail (Pin et al. 1992;Pin and Duvoisin 1995). Upon prolonged agonist addition, most GPCRs undergo decreased agonist responsiveness, a process known as desensitization. Recent studies have shown that desensitization of mGluR1a is G protein-coupled receptor kinase (GRK) dependent and that receptor phosphorylation by GRKs likely uncouples the GPCR from G-protein (Dale et al. 2000;Sallese et al. 2000). Following receptor G-protein uncoupling many GPCRs are removed from the cell surface by a process known as internalization (Krupnick and Benovic 1998). For some receptors, such as the b 2 -adrenoceptor, the binding of arrestin to receptor following GRK-dependent phosphorylation, serves to target the receptor to clathrin-coated pits for internalization (Goodman et al. 1996). Arrestins act as adaptors between the phosphorylated receptor and components of the endocytic machinery, such as AP-2 and clathrin, both of which are major components of clathrin-coated pits (Goodman et al. 1996...

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats

“…Another possibility is that the mechanism underlying the internalization of group I mGluRs after DHPG is different from that following glutamatemediated receptor activation. For instance, in vitro data have shown that application of glutamate versus the agonist quisqualate attract different arrestin proteins that aid in internalization of mGluR1a (Dale et al, 2001;Mundell et al, 2001). Thus, the mechanisms underlying group I mGluRs internalization appear to be quite complex and highly dependent on various factors including the source and pharmacological properties of the activating agonist.…”

“…In cell cultures and mice brain slices, mGluR1a and mGluR5 undergo agonist-stimulated internalization and endocytosis (Dale et al, 2001;Mundell et al, 2001) which, in some cases, was correlated with decreased group I mGluR-mediated physiological effects (Fourgeaud et al, 2004). However, there has been no in vivo study looking at changes in the trafficking of group I mGluRs following glutamate or receptor agonist stimulation in the mammalian brain.…”

Alsin Is a Rab5 and Rac1 Guanine Nucleotide Exchange Factor

“…Moreover, inhibition of caspases and cytochrome c release and overexpression of antiapoptotic proteins of the Bcl-2 family can prolong survival in these models (79,80,84). An overwhelming amount of evidence exists showing that ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, N-methyl-D-aspartate, and metabotropic (mGlu) glutamate receptors are cleared from the cell surface by endocytosis (85)(86)(87)(88)(89)(90)(91)(92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102). Rab5 activation is required for the endocytosis of many plasma membrane receptors.…”

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

“…13 Briefly, arrestintransfected cells were grown on poly-L-lysine-coated coverslips. Receptor distribution was assessed with a rhodamine-conjugated mouse monoclonal anti-HA Ab (1:100).…”