Ontogeny of CRLR and RAMPs in Myometrium

描述(申请人提供)：有效的平滑肌松弛分子 与子宫收缩活动的调节有关 在怀孕期间保持子宫静止。在缺席的情况下或 子宫内这些分子失衡，胎儿就会出生 为时过早。降钙素基因相关肽(CGRP)是最有效的平滑 已知的肌肉松弛多肽，也是参与 子宫静止。我们最近的研究表明，CGRP抑制子宫 妊娠期间的收缩，但不是在分娩期间，这可能是由于 1251-CGRP结合位点的变化。降钙素基因相关肽受体的研究进展 受阻于缺乏关于受体类型的明确数据 降钙素基因相关肽发挥作用。最近，有报道称降钙素 受体样受体(CRLR)与降钙素基因相关肽受体相关时发挥CGRP受体的作用 一种新的受体活性修饰蛋白(RAMP)1.1998年的这一发现 RAMPS改变CRLR结合的特异性开辟了一种新的范式 子宫降钙素基因相关肽的受体功能关系。我们有 初步证据表明CRLR和RAMP1在大鼠和大鼠脑内均有表达 人类的子宫。需要检验的总体假设是子宫反应性 TO-CGRP部分受CRLR和RAMPS差异表达的调节 这种表达是受激素调节的。当前 降钙素基因相关肽受体表达和功能的研究因 缺乏包括抗体和cDNA在内的试剂来探测受体成分 表情和功能。这项建议的目标是：1)确定 并合成了CRLR、RAMP1、RAMP2和RAMP3的独特多肽序列 大鼠和人，然后制备单克隆抗体进行免疫组织化学 研究，免疫印迹和研究激素调节的表达 受体，2)从大鼠和人中分离CRLR和RAMP1、2和3的cDNA 用于原位杂交和Northern分析的子宫肌层；3) 利用这些试剂确定CRLR和坡道的地形定位 大鼠和人子宫肌层的个体发育及随分娩时间的变化 研究它们的荷尔蒙调节。对发生的描述，个体发育 调节CRLR和子宫内的斜坡是关键的第一步 开始探索使用刺激或抑制的具体方法 受体作为产酵解的新的选择性手段。必要的第一步是 开发高质量试剂以协助这项研究并进一步用于 探索受体结构/功能关系。

DESCRIPTION (provided by applicant): 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.