Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop

Stephen S.G. Ferguson,Andy V. Babwah,Gurpreet K. Dhami,Rachel Sterne-Marr

doi:10.1074/jbc.m501650200

Stephen S.G. Ferguson, Andy V. Babwah + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m501650200

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Abstract

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

Highlights

Similar to most G protein-coupled receptors (GPCRs), Metabotropic glutamate receptors (mGluRs) activity is regulated by serine/threonine protein kinases [5,6,7,8,9]
Receptor/G protein interactions have been localized to the second intracellular loop (15, 26 –28), we further characterized GRK2 interactions with the second intracellular loop domain of mGluR1 by creating a series of four alanine scanning mutant GST fusion peptides that comprised the mGluR1 s intracellular loop flanked on each side by 5 amino acid residues from transmembrane domains 4 and 5 (Fig. 1C)
Because we had previously identified amino acid residue D527 in the GRK2 RH domain as critical for mediating GRK2 interactions with mGluR1b [24], we hypothesized that this acidic amino acid residue might associate with the three basic lysine residues localized within the second intracellular GSKKK motif

Summary

EXPERIMENTAL PROCEDURES

Materials— HEK293 human embryonic kidney cells were from American Type Culture Collection (Manassas, VA). For GST fusion proteins, mGluR1a PCR-generated sequences were cloned as GST-tagged EcoRI-XhoI products into the GST-prokaryotic expression vector pGEX-4T (GE Healthcare). GST-mGluR1a peptides bound to the matrix were washed extensively in PBS containing 0.3% Triton X-100. HEK293 cell lysates overexpressing GRK2 were prepared and cleared of cellular debris by centrifugation; 500 ␮g of total protein was used in each pull-down assay. Matrix-bound mGluR1a peptides and HEK293 cell lysates were incubated together and mixed overnight at 4 °C. Co-immunoprecipitation—The cells from 100-mm dishes were washed twice with ice-cold PBS and lysed in 400 ␮l of cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100 containing protease inhibitors, 20 ␮g/ml leupeptin, 20 ␮g/ml aprotonin, and 20 ␮g/ml phenylmethylsulfonyl fluoride). Statistical significance was determined by one-way analysis of variance with Tukey’s post hoc multiple comparison test

RESULTS

TABLE I

DISCUSSION