Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA).
The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.
Pula
Giordano
G
Department of Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. g.pula@bristol.ac.uk
Mundell
Stuart J
SJ
Roberts
Peter J
PJ
Kelly
Eamonn
E
D000410:Alanine Transaminase / Q000378:metabolism*; D000595:Amino Acid Sequence; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018094:Receptors, Metabotropic Glutamate / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; D000410:Alanine Transaminase