The role of neuropeptides in the brain and peripheral nervous system is being increasingly recognized. For example, in the pain sensory pathways bradykinin is strongly excitatory at the sensory endings; while such peptides as enkephalin, neurotensin, and salmon calcitonin (or related peptide) play a modulatory role in the central parts of the pathway. The overall goal of this project is to develop a reliable way of cloning the cDNAs and the genes that code for peptide receptors. The cDNAs could then be used to study how the levels of these receptors increase or decrease in disease states, or in response to drug treatment; and to obtain structural information about the receptors themselves, which would help in understanding how the peptides exert their effects. The difficulty in cloning cDNA for a peptide receptor is that it is in general present at very low levels in conventional cDNA libraries. An approach that has been used in the past is to enrich for rare sequences by hybridizing cDNA with RNA that does not contain the sequences, and removing the double-stranded hybrids. If the cDNA and the RNA are otherwise similar, this can produce a substantial enrichment. This project focusses on the bradykinin receptor, and uses subtraction of a cDNA library with a second cDNA library as a key step in the cloning strategy. The cloning strategy used in this project is in principle widely applicable for cell surface receptors in the nervous system. An application that would be of special interest is to clone the neuropeptide receptors that modulate transmission in central pain sensory pathways. The approach has other potential uses in comparing gene expression in different regions of the brain, at different developmental stages, or in normal and pathologically altered brain.
