Reducing conditions differentially affect the functional and structural properties of group-I and -II metabotropic glutamate receptors

We have examined the influence of reducing conditions on the activity of gr oup-I or -II metabotropic glutamate receptors. In cultured cerebellar granu le cells or in hippocampal slices, the reducing agent dithiothreitol (DTT) inhibited the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by group-I mGlu receptor agonists without affecting responses to norepinep hrine or carbamylcholine. Similarly, DTT reduced the increase in intracellu lar free Ca2+ induced by glutamate in HEK-293 cells expressing mGlu5 recept ors. Tn adult hippocampal slices, the selective group-II mGlu receptor agon ist, (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) had no e ffect per se on PPI hydrolysis, but potentiated the response to quisqualate . Although DTT substantially attenuated the action of quisqualate, it did n ot affect the potentiation by DCG-IV, suggesting that group-II mGlu recepto rs are resistant to extracellular reduction. Accordingly, DTT did not affec t the inhibition of forskolin-stimulated cAMP formation induced by maximall y effective concentrations of group-II mGlu receptor agonists in hippocampa l slices or in CHO cells expressing mGlu2 receptors. At structural level, D TT differentially affected the aggregation state of mGlu1a, -2/3 or -5 rece ptors. In immunoblots performed under non-reducing conditions, mGlu1a, -2/3 or -5 antibodies labeled exclusively a high-molecular weight band, corresp onding to receptor dimers, Under reducing conditions, mGlu1a or -5 receptor s were detected as monomers, whereas a large proportion of mGlu2/3 receptor s was still present in a dimeric form. We conclude that reducing conditions differentially influence the aggregation state of group-I and -II mGlu rec eptors and suggest that dimerization affects the functional activity of nat ive mGlu receptors. (C) 2000 Elsevier Science B.V. All rights reserved.