Potent smooth muscle relaxant molecules are implicated in the regulation of uterine contractile activity and maintenance of uterine quiescence during pregnancy. In the absence or imbalance of these molecules within the uterus, fetus will be delivered prematurely. Calcitonin gene-related peptide (CGRP) is the most potent smooth muscle relaxant peptide known, and is also a major component involved in uterine quiescence. Our recent studies show that CGRP inhibits uterine contractility during pregnancy, but not during labor and this may be due to changes in 1251-CGRP binding sites. Studies on receptors for CGRP have been hampered by the lack of clear data on the types of receptors through which CGRP exerts its effects. Recently, it has been reported that calcitonin receptor-like receptor (CRLR) functions as CGRP receptor when associated with a novel receptor activity modifying protein (RAMP)1. This discovery in 1998 of RAMPs modifying the specificity of binding of CRLR opened a new paradigm in receptor function relationships for CGRP effects in the uterus. We have preliminary evidence that both CRLR and RAMP1 are expressed in the rat and human uterus. The overall hypothesis to be tested is that uterine responsivity to CGRP is in part regulated by differential expression of CRLR and RAMPs in the myometrium and that this expression is hormonally regulated. Current investigations of CGRP receptor expression and function are hampered by the lack of reagents including antibodies and cDNAs to probe receptor component expression and function. The objectives of this proposal are to: 1) identify and synthesize unique peptide sequences for CRLR, RAMP1, RAMP2, and RAMP3 of rat and humans and then prepare monoclonal antibodies for immunohistochemical studies, Western blotting and to study hormonal regulation of expression of receptors, 2) isolate cDNAs for CRLR and RAMP1, 2, and 3 from rat and human myometrium for use in insitu hybridization and Northern analysis, and 3) utilize these reagents to determine topographic localization of CRLR and RAMPs in rat and human myometrium, determine their ontogeny and change with labor and study their hormonal regulation. Description of the occurrence, ontogeny and regulation of CRLR and RAMPs in the uterus is a crucial first step in beginning to explore the use of stimulation or inhibition of specific receptors as novel selective means of tocolysis. A necessary first step is the development of quality reagents to aid in this study and for further use in probing receptor structure/function relationships.
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