Isolation, characterisation, and chromosomal localisation of the mouse and human vasoactive intestinal peptide receptor type 2 genes

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been implicated in a wide range of functions in the central and peripheral nervous systems, including smooth muscle relaxation, the promotion of neuronal survival, modulation of the immune system and the control of embryonic growth. The VIP type 2 (VIP2) receptor is a seven-transmembrane G-protein coupled receptor belonging to the secretin/glucagon receptor subfamily, and is one of two such receptors cloned to date that bind both VIP and PACAP with high affinity. Studies of the distribution of the VIP2 receptor have provided some clues as to the possible functions of this receptor, and in particular have led to the proposal that this receptor may be largely responsible for mediating the neuroendocrine effects of VIP. However the absence of well characterised specific antagonists for the VIP2 receptor has proved to be a major obstacle to the delineation of the physiological role of this receptor. The work presented in this thesis has focused on the isolation and characterisation of mouse and human genomic clones encoding the VIP2 receptor, which should allow molecular genetic approaches to be used in future studies of the functions of this receptor. As a result of hybridisation screening of a mouse genomic A2001 library and a human genomic PI library, 6 bacteriophage clones that together span the entire coding region of the mouse VIP2 receptor gene (Vipr2), and a single PI clone that was shown to contain at least part of human VIP2 receptor gene (VIPR2), were isolated. Characterisation of the intron/exon structure of the mouse Vipr2 gene, achieved mainly by direct sequencing of A, DNA and PCR amplification of introns, revealed that the gene contained at least 12 introns, with sizes that range from 66 bp to at least 12 kb, and spans a minimum of 50 kb in total. Following the isolation of a 6 kb subclone which included the first exon and 5' sequence from the Vipr2 gene, 3 kb of the putative promoter region of the gene was sequenced. The proximal 5' region of the Vipr2 gene was found to be GC rich and CpG rich. No TATA box sequences were apparent within the region immediately upstream of the proposed transcription start site, but a computer-based search for possible transcription factor binding sites identified potential Spl and AP2 sites. Other potential regulatory sites that were found within the 3 kb sequence included possible binding sites for islet-1-like factors, the pituitary factor Pit1, cAMP responsive factors, serum response factor, interferon stimulated gene factor2, STAT factors, and factors that bind to the conserved lymphokine element 0. The chromosomal localisation of the VIP2 receptor gene in human and mouse was determined through collaborative work, in which the VIPR2 PI clone and a 4 kb subclone from the mouse gene were used as probes in fluorescence in situ hybridisation experiments. The mouse gene was mapped to the telomeric region of mouse chromosome 12 (12F2), and the human gene was mapped to 7q36.3. These results were interesting from two points of view: i) the genes localise to regions within the mouse and human genomes, that had not previously been recognised as being syntenic, and may therefore define a new region synteny, ii) the localisation of the human gene to 7q36.3 placed it within the previously defined minimal critical region for the brain developmental disorder holoprosencephaly type 3 (HPE3). However, subsequent, more detailed mapping of the position of the gene relative to the chromosomal deletions/rearrangements found in patients with HPE3, demonstrated that although one copy of the VIPR2 gene is deleted in some patients who have HPE3 and may contribute to the phenotype observed in these cases, it is not the gene responsible for HPE3.