Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization.
To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.
Mundell
Stuart J
SJ
Department of Pharmacology, School of Medical Sciences, University of Bristol, UK.
Pula
Giordano
G
Carswell
Katy
K
Roberts
Peter J
PJ
Kelly
Eamonn
E
D000595:Amino Acid Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism*; D018094:Receptors, Metabotropic Glutamate / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D018690:Excitatory Amino Acid Agonists; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; D018698:Glutamic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases