Elsevier

Neuropharmacology

Volume 36, Issue 2, February 1997, Pages 145-152
Neuropharmacology

Cloning, Distribution and Functional Expression of the Human mGlu6 Metabotropic Glutamate Receptor

https://doi.org/10.1016/S0028-3908(96)00172-4Get rights and content

Abstract

The cDNA encoding the human metabotropic glutamate receptor type 6 (hmGlu6) was isolated from a human retinal cDNA library. The deduced primary sequence (877 amino acids) of the hmGlu6 receptor was 93.5% identical to its rat counterpart and shared 69.8% sequence identity with the related hmGlu4 receptor clone (912 amino acids), isolated in parallel from a human brain cDNA library. In situ hybridization revealed that the hmGlu6 mRNA is highly expressed in cells located in the inner nuclear layer of the human retina, presumably bipolar neurons. Neither PCR analysis nor in situ hybridization could detect hmGlu6 mRNA in human brain. When stably expressed in Chinese hamster ovary cells (CHO-K1) the hmGlu6 receptor inhibited adenylate cyclase through a pertussis toxin-sensitive G-protein, and reduced forskolin-elevated cyclic adenosine monophosphate (cAMP) levels in response to agonists. The rank order of agonist potency was l(+)-2-amino-4-phosphonobutyric acid (l-AP4) > l-serine-O-phosphate > l-glutamate > quisqualate = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD). (2S,3S,4S)-α-(carboxycyclopropyl)-glycine (l-CCG-1) was a partial agonist at the hmGlu6 receptor, with a potency approaching that of l-serine-O-phosphate. © 1997 Published by Elsevier Science Ltd. All rights reserved.

Section snippets

Isolation of cloned receptor cDNAs

A human retinal cDNA library and a human whole brain library (5 × 105 plaques) (Clontech), both constructed in the bacteriophage λgt10, were screened for human mGlu receptor cDNAs. Fragments of the rat mGlu4 receptor cDNA (kindly donated by Professor Nakanishi, Kyoto, Japan), obtained by NcoI and KpnI/SphI digestions, were internally radiolabelled by random priming (Boehringer Mannheim) using [α-32P]dCTP (Amersham) to a specific activity of 2 × 109 c.p.m./μg and used as probes. Positively

RESULTS

The deduced amino acid sequence of hmGlu6 is presented in Fig. 1, in comparison to the rat mGlu6 receptor and the human mGlu4 receptor, cloned here in parallel. The hmGlu6 receptor is a protein of 877 residues which shows 93.5% sequence identity to the rat mGlu6 sequence. The similarity increases to 96.1% when conservative substitutions are allowed. The region of greatest diversity is found within the proposed signal peptide sequence. Other differences are scattered throughout the receptor

DISCUSSION

We have cloned the human mGlu6 metabotropic glutamate receptor, which shares high sequence identity (93.5%) with its rat homologue, and reasonably high identity (69.8%) with the related human mGlu4 receptor, cloned in parallel (Fig. 1). The serine and threonine residues essential for high affinity for l-glutamate (O'Hara et al., 1993) are conserved (Fig. 1), as are many cysteine residues found in all mGlu receptors cloned to date (see Pin and Duvoisin, 1995). These cysteine residues presumably

Acknowledgements

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

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