We have recently discovered and characterized a novel hormone, intermedin/adrenomedullin 2, belonging to the CGRP/adrenomedullin peptide family. Functional studies showed that intermedin signals through a G protein-coupled receptor, calcitonin receptor-like receptor (CLR). CLR is known to mediate the action of CGRP and adrenomedullin, but efficient signaling by these hormones requires one of three coreceptors known as receptor activity-modifying proteins (RAMP1, 2, and 3). CGRP preferentially signals through cells expressing RAMP1 and CLR whereas adrenomedullin exhibits greater potency on cells expressing RAMP2 and CLR, or RAMPS and CLR. In contrast, the newly identified intermedin preferentially activates RAMP1- and RAMP-3 mediated CLR signaling. Therefore, intermedin could regulate novel physiological processes or those previously shown to be regulated by CGRP or adrenomedullin. Furthermore, we have demonstrated that intermedin exhibits potent cardiovascular effects and represents an estrogen-dependent prolactin- releasing hormone in the pituitary. Our preliminary data shows that, 1) a tethered CGRP-RAMP1 chimera constitutively activates CLR in the absence of a ligand, and 2) direct interaction of the tethered CGRP- RAMP1 chimera with CLR results in the formation of a stable complex essential for G protein coupling. Thus, we hypothesize that activation of CLR signaling by CGRP family peptides could involve three sequential steps that lead to the formation of a trimeric complex consisting of the ligand, RAMP, and CLR. Because selective interactions between CGRP family peptides with RAMP proteins are crucial in the selective activation of CLR, in Specific Aim 1 we propose to study the structural-functional characteristics of the CGRP family peptides and generate RAMP-selective agonists and antagonists.
In Specific Aim 2, we will investigate the role of ligand-RAMP interactions in the formation of a trimeric ligand-RAMP-CLR complex for signaling using tethered ligand-RAMP fusion proteins.
In Specific Aim 3, taking advantage of findings that the tethered CGRP-RAMP1 chimera constitutively activates CLR, we will generate and characterize transgenic mice with the tethered CGRP-RAMP1 chimera using a knock-in strategy. Because the CGRP-RAMP1 transgene is flanked by LoxP sites, we also can generate conditional RAMP1-deficient mice for future studies. Altogether, this proposal aims to answer two of the critical questions in the study of CGRP family peptides: how receptor specificity is determined and what are the physiological roles of individual RAMPs in vivo. By dissecting the molecular mechanisms underling ligand-receptor interactions and specifically investigating the RAMP1- mediated CLR signaling in transgenic mice, these experiments could facilitate the determination of functional motifs in both ligands and RAMPs for CLR activation as well as illustrate the physiological significance of individual RAMP proteins in diverse physiological processes and cardiovascular diseases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Molecular and Cellular Endocrinology Study Section (MCE)
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Silva, Corinne M
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Stanford University
Obstetrics & Gynecology
Schools of Medicine
United States
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Chang, Chia Lin; Cai, James J; Huang, Shang Yu et al. (2014) Adaptive human CDKAL1 variants underlie hormonal response variations at the enteroinsular axis. PLoS One 9:e105410
Chang, Chia Lin; Semyonov, Jenia; Cheng, Po Jen et al. (2013) Widespread divergence of the CEACAM/PSG genes in vertebrates and humans suggests sensitivity to selection. PLoS One 8:e61701
Chang, Chia Lin; Hsu, Sheau Yu Teddy (2013) Roles of CLR/RAMP receptor signaling in reproduction and development. Curr Protein Pept Sci 14:393-406
Chang, Chia Lin; Cai, James J; Lo, Chiening et al. (2011) Adaptive selection of an incretin gene in Eurasian populations. Genome Res 21:21-32
Chang, Chia Lin; Cai, James J; Cheng, Po Jen et al. (2011) Identification of metabolic modifiers that underlie phenotypic variations in energy-balance regulation. Diabetes 60:726-34
Chang, Chia Lin; Wang, Hsin-Shih; Soong, Yung-Kuei et al. (2011) Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2. J Biol Chem 286:43193-203
Chang, Chia Lin; Park, Jae-Il; Hsu, Sheau Yu Teddy (2010) Activation of calcitonin receptor and calcitonin receptor-like receptor by membrane-anchored ligands. J Biol Chem 285:1075-80
Pfeil, Uwe; Aslam, Muhammad; Paddenberg, Renate et al. (2009) Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability. Am J Physiol Lung Cell Mol Physiol 297:L837-45
Semyonov, Jenia; Park, Jae-Il; Chang, Chia Lin et al. (2008) GPCR genes are preferentially retained after whole genome duplication. PLoS One 3:e1903
Park, Jae-Il; Semyonov, Jenia; Chang, Chia Lin et al. (2008) Origin of INSL3-mediated testicular descent in therian mammals. Genome Res 18:974-85