Pergamon PII: SOO28-3908(97)00078-6 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Neuropharmacology , Vol. 36, No. I, pp. 917-931, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028-3908/97 $17.00 + 0.00 Cloning and Stable Expression of the mGluRlb Subtype of Human Metabotropic Receptors and Pharmacological Comparison with the mGluR5a Subtype F. F. LIN,l M. VARNEY,’ A. I. SACAAN,’ C. JACHEC,’ L. P. DAGGETT,’ S. RAO,’ P. FLOR, R. KUHN,2 J. A. KERNER,3 D. STANDAERT; A. B. YOUNG3 and G. VELICELEBI’* ‘SIBIA Neurosciences Inc., 505 Coast Blvd. South, La Jolla, CA 92037, U.S.A.; ‘Novartis Phanna Inc., CNS Research, CH-4002 Basel, Switzerland; and 3Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, U.S.A. (Accepted I7 March 1997) Summary-We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype lb (hmGluRlb). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 rnRNA in the dentate gyms of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluRlb in L(tl-) mouse fibroblast and Chinese hamster ovary (CHO-dhft-) cells. In both expression systems, agonist activation of hmGluRlb stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca*‘]J, and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (lS,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluRlb and hmGluRSa, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate 2 (RS)-3,5-DHPG > (lS,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluRlb. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca*‘]i in both hmGluRlb- and hmGluR5a-expressing cells. (S)4CPG and (S)- 4C3HPG both inhibited agonist responses only in hmGluRlb-expressing cells. However, in hmGluRSa- expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and caellular level and as screening targets for identification of subtype-selective agonists or antagonists. 0 1997 Elsevier Science Ltd. Keywords-Cloning, distribution, functional expression, pharmacological profiling. Glutamate is the major excitatory neurotransmitter in the distinct subtype-selective anatomical distribution pat- mammalian central nervous system and plays an terns (for review, see Hollmann and Heinemann, 1994). important role in modulation of synaptic transmission The functional and pharmacological properties of and plasticity. The effects of glutamate are mediated via the ionotropic f’+methyl-D-aspartate (NMDA) and non- recombinant metabotropic glutamate receptors have been NMDA receptors and metabotropic glutamate receptors studied in Xenopus oocytes and mammalian cells (Abe et (mGluRs) (for review, see Nakanishi, 1992). Activation al., 1992; Brabet et al., 1995; Desai et al., 1995; Flor et of glutamate receptors is also involved in some al., 1995a,b; Hayashi et al., 1994; Houamed et al., 1991; neuropathological conditions, such as ischemia and Joly et aZ., 1995; Kingston et al., 1995; Laurie et al., epilepsy (Choi, 1988; Collingridge and Singer, 1990; 1996; Masu et al., 1991; Minakami et al., 1994; Dingledine et al., 1990; Meldrum and Garthwaite, 1990; Nakajima et al., 1993; Okamoto et al., 1994; Pin et al., Monaghan et al., 1989; Schoepp et al., 1990; Szatkowski 1992; Saugstad et “*’ 1994; Tanabe et al., 1992; and Attwell, 1994). Glutamate receptors are ubiquitous in Thomsen et al., 1994). The results have revealed that in the mammalian central nervous system and display these expression systems the class I mGluRs (mGluR1 and mGluR5) are functionally coupled to stimulation of InsP formation and mobilization of Ca2’ from intracel- *To whom correspondensce should be addressed. Tel: (619) lular stores, whereas class II mGluRs (mGluR2 and 452-5892 Ext. 227; Fax: (619) 452-9279. mGluR3) and class III mGluRs (mGluR4, mGluR6, 917