A flanking gene problem leads to the discovery of a Gprc5b splice variant predominantly expressed in C57Bl/6J mouse brain and in maturing neurons.
BACKGROUND
Gprc5b, a retinoic acid-inducible orphan G protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses.


RESULTS
We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3' terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line.


CONCLUSIONS
Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences.
Cool
Bethany H
BH
Department of Pathology, University of Washington, Seattle, Washington, USA.
Chan
Guy C-K
GC
Lee
Lin
L
Oshima
Junko
J
Martin
George M
GM
Hu
Qubai
Q
D000818:Animals; D001921:Brain / Q000254:growth & development / Q000378:metabolism*; D001923:Brain Chemistry; D020869:Gene Expression Profiling; D007178:Inbreeding; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000032:analysis; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000235:genetics*
C410799:GPRC5B protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled