Molecular characterization of chicken glutamate receptor, metabotropic1 (GRM 1)

Abstract

Glutamate is the most abundant excitatory neurotransmitter in the mammalian nervous system. Ionotropic glutamate receptors used to be the only type of glutamate receptors, bringing about essential functions including synaptic transmissions. Since 1991, eight metabotropic glutamate receptors have been discovered. Belonging to the subfamily C of G protein-coupled receptor (GPCR) superfamily, these receptors have unique structural features. They couple to their own specific G proteins and transduce signals via pathways not recognized in other subfamilies. To date, little information on these receptors have been revealed in mammals, and even less is known about them in non-mammalian species including chicken.

In the present study, various cDNAs of the chicken glutamate receptor, metabotropic 1 (GRM1) as well as its splice variants were cloned from adult brain tissue. At least 11 exons were identified in the chicken (c-) GRM1 gene, in which the alternative usage of exons and splice acceptor sites results in at least three variants, namely cGRM1a, cGRM1b and cGRM1f. The predicted coding regions of cGRM1a, cGRM1b and cGRM1f are 3459 base pairs (bp), 2736 bp and 2697 bp in length, which were deduced to encode receptor peptides of 1152 amino acids (aa), 911 aa and 898 aa, respectively. The predicted cGRM1a peptide shows high amino acid sequence identities (87.5% to 88%) to its counterparts in humans, rats, mice, chimpanzees and cattle. cGRM1b transcript differs from cGRM1a transcript by inclusion of two additional exons (7b and 7c), which contains a premature stop codon and results in its shorter C-terminal tail. cGRM1f is a novel splice variant that lacks exon 7b and is 13 aa shorter than cGRM1b. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the transcripts of cGRM1a, cGRM1b and cGRM1f were preferentially expressed in adult chicken brains, in which cGRM1f mRNA was additionally identified in pituitary, lungs and gonads. Functional assay demonstrated that cGRM1a and cGRM1b receptors, expressed in Chinese hamster ovary cells, were induced by glutamate in dose-dependent manners via the Fura-2 dye calcium assays. In addition, dual luciferase reporter assays suggested that cGRM1a and cGRM1b receptors have no significant effects on the activation of cAMP/PKA and MAPK/ERK signaling pathways upon glutamate treatment.

Taken together, the present study has provided the first step in understanding the possible roles of GRM1 in chickens.