Although agonists bind directly in the heptahelical domain (HD) of most class-I rhodopsin-like G protein coupled receptors (GPCRs), class-III agonists bind in the extracellular domain of their receptors. extracellular domain name. This obtaining illustrates that, like rhodopsin-like receptors, the HD of mGluRs can constitutively couple to G proteins and be negatively and positively regulated by ligands. These data show that this HD of mGluRs behave like any other class-I GPCRs in terms of G protein coupling and regulation INK 128 inhibitor database by various types of ligands. = 8), MPEP also inhibited 42.8 1.5% (= 3) and 46.7 15.3% (= 7) of the basal IP formation measured in cells expressing 5 and 5, respectively (Fig. 2= 3, not shown) or 5 (10.4 6.6 nM, = 7) similar to that decided on mGlu5 (8.1 4.3 nM, = 8) (Fig. 2= 9) to 17.4 4.4 M(= 4) in the absence and presence of 100 M DFB, respectively] (Fig. 3= 5) and 13.1 5.5 nM (= 3) in the absence and presence of DFB, respectively, data not illustrated]. The maximal effect of glutamate was, however, not altered by DFB. As shown in Fig. 3= 6)(Fig. 4 em A /em ), INK 128 inhibitor database identical to that measured for the potentiating effect on the wild-type receptor (Fig. 3 em A /em ). A similar effect was obtained with 5 (data not shown). DFB was also able to induce a Ca2+ transmission in cells expressing 5 or 5 (Fig. 4 em B /em ), with EC50 values of 20.2 1.7 M and 19.6 1.2 M, respectively. These data Met show that, whereas DFB is usually a clear positive allosteric modulator devoid of agonist activity on mGlu5, it functions as an agonist around the truncated INK 128 inhibitor database receptors. Open in a separate windows Fig. 4. Direct activation of 5 by the positive allosteric regulator DFB. ( em A /em ) Effect of increasing doses of DFB on 5. DFB dose-dependently activates the truncated receptor 5. The curve has been normalized such that the basal response is usually zero and the maximum is usually 100%. ( em Inset /em ) IP formation (% above the basal) was induced by DFB only in cells expressing 5 and not in cells expressing mGlu5. ( em B /em ) Direct activation of 5 and 5 by 100 M DFB as revealed by intracellular Ca2+ measurement with Fluo-4. ( em C /em ) Activity of mGlu5 (squares) and 5 (circles) as a function of their membrane expression. Cells were transfected with increasing amounts of cDNA coding for these receptors, and surface expression of mGlu5 and 5 was measured by ELISA on intact cells. Basal (open symbols) and glutamate-(1 mM) or DFB-(1 mM) (packed symbols) induced IP formation was measured in parallel. DFB-Induced Activity of 5 Is usually Close to the Agonist-Induced Activity of mGlu5. To compare the activities of mGlu5 and 5, cell surface expression and basal and agonist-induced IP production were measured in cells transfected with numerous amounts of plasmid DNA. Both basal and glutamate-induced IP productions were directly proportional to the number of receptors at the cell surface, the slope of the correlation lines being indicative of the specific (amount of IP produced per receptor) basal and glutamate-induced activity of the receptor (Fig. 4 em C /em ). When the same analysis was performed with 5 in the absence and presence of DFB, the same correlation lines were obtained, showing that the specific constitutive activity of 5 is similar to that of mGlu5, and that DFB-induced activity of 5 is similar to the glutamate-induced activity of the wild-type receptor (Fig. 4 em C /em ). Inhibition of the DFB-Induced Response of 5 by MPEP. Because both DFB and MPEP were found to act on 5, we examined whether MPEP could inhibit the action of DFB. As shown in Fig. 5, MPEP inhibited the effect of DFB on IP production or increase in intracellular Ca2+ in cells expressing 5. However, even at high concentration, MPEP only partly inhibited the effect of DFB on 5. This obtaining indicates a complex conversation between MPEP and DFB binding sites, in agreement with the partial inhibition of [3H]methoxyPEPy (an analog of MPEP) binding by DFB around the wild-type receptor (31). Open in a separate windows Fig. 5. MPEP inhibits partially DFB-induced activity on 5. ( em A /em ) Effect of 10, 30, and 100 nM MPEP on IP production induced by 300 M DFB in cells expressing 5. ( em B /em ) Effect of 10 and 100 nM MPEP on intracellular Ca2+ release induced by 300 M DFB on cells expressing 5. Conversation The present study demonstrates that mGlu5 HD can activate PLC in the absence of.