Abstract
The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.
Highlights
The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1␣, triggers intracellular calcium release; mGluR5 activation is unique in that it elicits Ca2؉ oscillations
We showed that the analogous serine in mGluR1, Ser-853, is not phosphorylated due to an adjacent aspartic acid residue but can be phosphorylated when the adjacent amino acid is mutated to a permissive residue
protein kinase C (PKC) is an important modulator of Metabotropic glutamate receptors (mGluRs) function [10, 13,14,15,16,17]; the biochemical analyses characterizing the direct phosphorylation of mGluRs by PKC have been lacking
Summary
Vol 280, No 27, Issue of July 8, pp. 25409 –25415, 2005 Printed in U.S.A. Protein Kinase C Phosphorylation of the Metabotropic Glutamate Receptor mGluR5 on Serine 839 Regulates Ca2؉ Oscillations*. Metabotropic glutamate receptors (mGluRs) play important roles throughout the nervous system, including the activation of ion channels and the regulation of synaptic plasticity [1]. They have been implicated in a variety of neurological diseases [2,3,4,5]. A previous study [10] demonstrated that a short stretch of amino acids in the C terminus of mGluR5 was the critical determinant regulating Ca2ϩ oscillations, the identity of a single amino acid residue, Thr-840. We demonstrated that Thr-840 is not a PKC substrate but only plays a permissive role for PKC phosphorylation of the adjacent amino acid Ser-839 It is the PKC phosphorylation of Ser-839 that regulates mGluR5-elicited Ca2ϩ oscillations. Our data clearly demonstrate that this is because of the differential effect these residues have on the PKC phosphorylation of a conserved adjacent serine, not because of the direct PKC phosphorylation of Thr-840 on mGluR5
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