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Molecular cloning of a human putative 7-transmembrane segment (7TMS) receptor

University of British Columbia

Seven transmembrane-segment (7TMS) receptors are highly homologous proteins that mediate a variety of cell functions through the interaction with G-proteins. They are important mediators in the response to neurotransmitters, hormones and cytokines. Moreover, two of these receptors have been found to be proto-oncogenes or to possess transforming activity. During the immune response they are involved in inflammation, producing and modulating this process through the interaction with ligands that are primarily chemotactic and activating factors. A family of such ligands has already been characterized and their receptors, when known, were all of the 7TMS type. To further investigate this category of proteins in cells involved in immunity a group of oligonucleotides was patterned on predicted conserved areas of chemotactic 7TMS receptors, and also on divergent non-homologous sequences. To facilitate subcloning procedures restriction sites where engineered at the 5'-end of the primers. Human monocyte mRNA was converted to single strand cDNA by antisense priming and reverse transcription. This cDNA was used as a template for the in vitro enzymatic amplification by the polymerase chain reaction (PCR) with upstream and downstream primers. The amplified cDNA (388 bp) was cloned in plasmid Bluescript and sequenced by the chain termination method. Homology searches for the sequence revealed that the fragment belonged to the 7TMS superfamily. The cDNA was radiolabeled and used to screen a human fetal spleen library constructed in the ƛ-phage system. A clone was isolated that when sequenced revealed a complete open reading frame for a 352 residue protein. This polypeptide was found to be a human homologue of a neuropeptide receptor: neuropeptide tyrosine (NPY) subtype 3. Apart from having the typical topology of the 7TMS superfamily it also displayed a high degree of homology to chemotactic factor receptors. Northern blot analysis demonstrated that this protein is expressed in locations outside the nervous system like spleen and thymus.

Thesis/Dissertation

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2008-12-15

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http://hdl.handle.net/2429/2911

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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