@Pergamon

Neuropharmacology,

i+

Cloning, Distribution and Functional Expression of theHuman mGlu6 Metabotropic Glutamate Receptor

D.

NovartisPharmaInc.,Basel, Switzerland

(Accepted29 October 1996)

Summary-The cDNA 6a

shared69.870sequenceidentitywith the related hmGluAreceptorclone (912 amino acids), isolated in parallel from a human brain cDNA library.In situ

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Keyword%Metabotropic glutamate receptor types 4 and 6 (mGlud and mGlus), human, retina, Chinesehamster ovary cells, cAMP.

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*To whom correspondence should be addressed at: NovartisPharma Inc., S-386-809,CH-4002,Basle, SwitzerlandFax:061-324-5524; e-mail: bernd.sommer@Pharma-Novartis.corn.

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Human mGlu6receptor 147

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Fig. 1. Comparisonof the deducedamino acid sequencesfor the humanand rat mGlu6andhumanmGlu4receptors(standardone-lettercode).The humanmGlu6sequenceis givenin full. Wherethe sequenceof the othersdiffer,theamino acid is given. Numbers,indicate amino acid positions of the hmGlu6sequence. The putative N-terminalsignal peptide is underlined. Putative transmembrane domains I–VII are boxed. (+), proposed N-linkedglycosylationsites; (0), serine and threonineresiduesknownto be essential for glutamate affinity;(*), cysteines

conservedin all cloned mGlu receptors.

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of hmGh.16 isthe rat mGlU6

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148 D. J. Laurie et al.

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Fig. 2. PCR analysis of the distributionof the mRNA encodingthe hmGluGreceptor. Samples were run on a 1% agarose geland visualizedby ethidiumbromide stainingunder UV light. A804 bp band was predicted. Lanes: 1, cerebellum; 2, hippo-campus;3, cortex;4, retina; M, molecularweight marker?(basepairs; 19300, 7550, 6210, 4520, 3470, 2700, 1870, 1500, 920

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DISCUSSION

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Fig. 3. Distributionof the hybridisationsignal for the hmGlucreceptormRNAin humanretina. (A) Giemsastained section of human retina. L, lumen; R, retina; S, sclera. (B) Znsitu hybridisationsignal on X-ray film. (C)Competition control (100-foldexcess of unlabeled probe). Scale bar: 100pm. (D) Photomicrographof Giemsastained human retina dipped in photographic emulsion (darkfieldoptics). Scale bar: 10 pm. GCL, granule celllayer; IPL, inner plexiformlayer; INL, inner nuclear layer; OPL, outer plexiformlayer; ONL, outer nuclear layer;

PL, photoreceptorlayer.

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Fig. 4. T$e effect of various agonists on forskolin-elevatedcAMP levels in CHOihmG6.14cells. A typical experiment isshown. I@ults are expressed as a percentage of the forskolin(10 pM)-elevated cAMP level. (A), L-serine-O-phosphate; (+), L-glutamate; (0), (1S,3R)-ACPD; (0),quisqualate; (*), effects of L-glutamate after overnightpretreatment with pertussis toxin (100 rig/ml).Block indicates

level of cAMP in saline-treatedcontrols.

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authors are indebted to Mr A.Wanner for excellent sequencing, and Mr I. Bobirnac forquality cAMP measurements.

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