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 group-I or -II metabotropic glutamate receptors. In cultured cerebellar granule 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 norepinephrine or carbamylcholine. Similarly, DTT reduced the increase in intracellular free Ca(2+) induced by glutamate in HEK-293 cells expressing mGlu5 receptors. In adult hippocampal slices, the selective group-II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) had no effect per se on PPI hydrolysis, but potentiated the response to quisqualate. Although DTT substantially attenuated the action of quisqualate, it did not affect the potentiation by DCG-IV, suggesting that group-II mGlu receptors are resistant to extracellular reduction. Accordingly, DTT did not affect the inhibition of forskolin-stimulated cAMP formation induced by maximally effective concentrations of group-II mGlu receptor agonists in hippocampal slices or in CHO cells expressing mGlu2 receptors. At structural level, DTT differentially affected the aggregation state of mGlu1a, -2/3 or -5 receptors. In immunoblots performed under non-reducing conditions, mGlu1a, -2/3 or -5 antibodies labeled exclusively a high-molecular weight band, corresponding to receptor dimers. Under reducing conditions, mGlu1a or -5 receptors were detected as monomers, whereas a large proportion of mGlu2/3 receptors was still present in a dimeric form. We conclude that reducing conditions differentially influence the aggregation state of group-I and -II mGlu receptors and suggest that dimerization affects the functional activity of native mGlu receptors.
Copani
A
A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Catania, Viale A. Doria, 6, 95125, Catania, Italy.
Romano
C
C
Di Giorgi Gerevini
V
V
Nicosia
A
A
Casabona
G
G
Storto
M
M
Mutel
V
V
Nicoletti
F
F
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology; D018679:Cholinergic Agonists / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D003521:Cyclopropanes / Q000494:pharmacology; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007668:Kidney / Q000166:cytology; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D016318:Quisqualic Acid / Q000494:pharmacology; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000600:Amino Acids, Dicarboxylic; D000927:Anticonvulsants; D018679:Cholinergic Agonists; D003521:Cyclopropanes; D018690:Excitatory Amino Acid Agonists; D018696:Neuroprotective Agents; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2; C104077:metabotropic glutamate receptor type 1; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; C082313:2-(2,3-dicarboxycyclopropyl)glycine; D005576:Colforsin; C035261:(alpha-carboxycyclopropyl)glycine; D016318:Quisqualic Acid; D002217:Carbachol; D000242:Cyclic AMP; D004229:Dithiothreitol; D005998:Glycine