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

At present, little is known regarding the mechanism of metabotropic glutama te receptor (mGluR) trafficking. To facilitate this characterization we ins erted a haemagglutinin (HA) epitope tag in the extracellular N-terminal dom ain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transient ly transfected with HA-mGluR1a, the epitope-tagged receptor was primarily l ocalized to the cell surface prior to agonist stimulation. Following stimul ation with glutamate (10 muM; 30 min) the HA-mGluR1a underwent internalizat ion to endosomes. Further quantification of receptor internalization was pr ovided by ELISA experiments which showed rapid agonist-induced internalizat ion of the HA-mGIuR1a. To determine whether agonist-induced mGluR1a interna lization is an arrestin- and dynamin-dependent process, cells were cotransf ected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-4 18). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 muM; 30 min) HA-mGluR1a internali zation. In addition, wild-type arrestin-2-green fluorescent protein (arrest in-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cy tosol to membrane in HEK293 cells coexpressing HA-mGIuR1a. Taken together o ur observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.