Vol. 58, Issue 2, 352-360, August 2000

Complex Involvement of Pertussis Toxin-Sensitive G Proteins in the Regulation of Type 1 Metabotropic Glutamate Receptor Signaling in Baby Hamster Kidney Cells

Emmanuel Hermans, Ruth Saunders, Julie V. Selkirk, Rajendra Mistry, Stefan R. Nahorski, and R. A. John Challiss

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (E.H., R.S., J.V.S., R.M., S.R.N., R.A.J.C.); and Laboratoire de Pharmacologie, Université Catholique de Louvain, Brussels, Belgium (E.H.)

Previously, we demonstrated that the coupling of the metabotropic glutamate receptor mGlu1 to phosphoinositide hydrolysis is enhanced by pertussis toxin (PTX) in stably transfected baby hamster kidney cells (BHK). Here, we show that the PTX effect on agonist-stimulated [³H]inositol phosphate accumulation can be resolved into two components: an immediate increase in agonist potency, and a more slowly developing increase in the magnitude of the response observed at maximally effective agonist concentrations. Using G_q/11- and G_i/o-selective antibodies to immunoprecipitate [³⁵S]guanosine-5'-O-(3-thio)triphosphate-bound G proteins, we also show that agonist stimulation of mGlu1 in BHK membranes increases specific [³⁵S]guanosine-5'-O-(3-thio)triphosphate binding to both G_q/11 and G_i/o proteins. Preincubation of BHK-mGlu1 with L-glutamate (300 µM) results in a progressive loss (60% in 30 min) of L-quisqualate-induced [³H]inositol phosphate accumulation (without a change in potency), providing evidence for agonist-induced receptor desensitization. Although such desensitization of mGlu receptor signaling was mimicked by a phorbol ester, agonist-induced phosphorylation of the receptor was not observed and protein kinase C inhibition by Ro 31-8220 did not prevent L-glutamate-mediated desensitization. In contrast, PTX treatment of the cells almost completely prevented L-glutamate-mediated desensitization. Together, these data provide evidence for a multifunctional coupling of mGlu1 to different types of G proteins, including PTX-sensitive G_i-type G proteins. The latter are involved in the negative control of phospholipase C activity while also influencing the rate of desensitization of the mGlu1 receptor.