Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Abstract

SUMMARYThe regulation of phosphoinositide hydrolysis by the type 1␣ metabotropic glutamate receptor (mGluR1␣) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S,3R-dicarboxylic acid resulted in a marked accumulation of 3 H]InsP 1 responses were similar in control and PTXtreated BHK-mGluR1␣ cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (G i/o ) and PTX-insensitive stimulatory (G q/11 ) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1␣-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.The recent cloning of eight subtypes of mGluR has not only opened up new avenues for exploration of the central actions of this excitatory neurotransmitter but also expanded the potential to target drugs against specific receptor-mediated actions (1-3). Recently, novel synthetic glutamate analogues have been developed as ligands at different mGluRs, and of particular significance has been the development of competitive antagonists (4 -7) and their use in identifying the involvement and roles of different mGluR subtypes in fundamental mechanisms such as long term potentiation and long term depression (8 -12).The mGluRs form a distinct branch of the G protein-coupled receptor superfamily, sharing topological organization but little sequence homology with other G protein-coupled receptors. Sequence homology and pharmacological profiling have allowed three subgroups of mGluRs, termed I, II, and III, to be described (2, 3). Group II mGluRs (types 2 and 3) and group III MGluRs (types 4, 6, 7 and 8) both couple to G proteins of the G i/o family to inhibit adenylyl cyclase or modulate ion channel activities (2, 3); in contrast, group I mGluRs (types 1 and 5) activate PIC with the subsequent generation of the second messengers Ins(1,4,5)P 3 and diacylglycerol (2, 3, 13-17). The G protein or proteins responsible for coupling group I mGluRs to PIC have been the subject of some debate. Thus, although the phosphoinositide responses elicited by agonist stimulation of mGluR5 and the mGluR1␤ splice variant seem to be little affected by PTX treatment (14,15), the response to mGluR1␣ activation is substantially

Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes

“…The coupling of group I mGluR to PI-PLC, as demonstrated by inositol phosphate measurements, has been shown in many systems (Abe et al, 1992;Aramori & Nakanishi, 1992;Carruthers et al, 1997;Wakelam, 1998). Here we show that there was no detectable increase in IP 3 in synaptosomes within 15 s of receptor stimulation, a time point at which we can measure an elevated IP 3 response to carbachol in cerebellar granule cells (del Rio et al, 1998).…”

“…PI-PLC activation is conventionally measured as an increase in IP 3 or DAG concentration, the two products of PIP 2 hydrolysis by PLC. mGluRs activate PI turnover with fast kinetics (Aramori & Nakanishi, 1992;Carruthers et al, 1997), therefore, we measured IP 3 after 15 s. However, we could not detect any change in IP 3 levels by receptor stimulation with either 1 mM quisqualate (a non-selective mGluR agonist) or 100 mM 1S,3R-ACPD (Figure 4). This method however has been successfully employed to demonstrate an elevation of IP 3 in carbachol stimulated cerebellar granule cells (del Rio et al, 1998).…”

Effects of Human Type 1α Metabotropic Glutamate Receptor Expression Level on Phosphoinositide and Ca2+ Signalling in an Inducible Cell Expression System

“…lA). However, preaddition of GPT (3 U ml~' ) and pyruvate (3 mM) to decrease the concentration of glutamate in the incubation medium (Desai et al, 1995;Carruthers et al, 1997) was found to inhibit completely this basal increase, suggesting that endogenous glutamate released from the cells can have a stimulatory effect when the hmGlula receptor is expressed at high levels (data not shown). Furthermore, the increased basal 3H-InsP level measured in IPTG-induced cells was also found to be inhibited by the mGlu receptor antagonist 4-C3HPG (used at 300 ‚uM).…”

“…Determination of basal and agonist-induced accumulation of 3H-InsP in CHO-lac-hmGlula cells was performed as previously described (Carruthers et al, 1997). When required, IPTG was added directly to the culture medium for the times indicated.…”

Metabotropic Glutamate 1 Receptor: Current Concepts and Perspectives

“…However, this coupling applies to rat mGlu1 receptors, whereas human mGlu1␣ and -␤ receptors seem to be coupled to PI hydrolysis with equal efficacy (Stephan et al, 1996). The mGlu1␣ receptor but not the mGlu1␤ or mGlu1␥ receptor is constitutively active in stimulating PI hydrolysis; i.e., it is active in the absence of an orthosteric agonist, in transfected LLC-PK1, HEK-293, or BHK cells (Joly et al, 1995;Prézeau et al, 1996;Carruthers et al, 1997;Mary et al, 1997;Hiltscher et al, 1998). This constitutive activity is not prevented by orthosteric mGlu1 receptor antagonists, which reduce the response to applied glutamate, but not the basal activity; hence, this activity is not triggered by endogenous glutamate present in the culture medium (Prézeau et al, 1996).…”