When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptorlike receptor (CRLR) can function as a calcitonin generelated peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86 -92 significantly attenuated high-affinity 125 I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59 -65 had a similar effect. There is little sequence identity between hRAMP3 residues 59 -65 and hRAMP2 residues 86 -92; moreover, substituting alanine for of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR. CGRP1 and AM belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators (1, 2). Although they share very little sequence identity, both proteins contain ring structures comprised of six or seven amino acids linked by a disulfide bridge and an amidated C terminus required for biological activity (3). Both peptides and their specific or common binding sites are widely distributed among peripheral tissues and in the central nervous system and mediate a wide variety of biological effects (4 -6).In 1998, McLatchie et al. (7) identified and cloned the RAMP family of accessory proteins that includes three isoforms (RAMP1, RAMP2, and RAMP3) and serves to transport the CRLR to the cell surface, thereby forming functional CGRP and AM receptors. RAMPs consist of ϳ160 amino acids and possess a large extracellular N-terminal domain, a single transmembrane-spanning domain, and a short cytoplasmic domain. Coexpression of RAMP1 with CRLR leads to both proteins being presented at the plasma membrane as a CGRP receptor, while co-expression of RAMP2 or -3 allows CRLR to function as an AM receptor (7-10). Interestingly, although RAMP2 and -3 share only 30% sequence identity and differ in their tissue distributions, they generate essentially equivalent AM receptors when co-expressed with CRLR in HEK293T cells (9).It was initially believed that mature glycosylation of CRLR in the presence of RAMP1 determined CGRP specificity, while core glycosylation in the presence of RAMP2 or -3 determined AM specificity (7, 9). However, unlike in mammalian cells, co-expressi...

Adrenomedullin (AM) and atrial and brain natriuretic peptides (ANP and BNP) exert vasodilator and natriuretic actions and are thought to share roles in counteracting the progression of hypertension or heart failure as circulating or locally-acting hormones. However, little data is available with regard to their roles in subjects who have no apparent cardiovascular diseases. The present study was carried out to identify the factors that affect plasma levels of AM, ANP and BNP in the general population. We measured the plasma levels of AM, ANP and BNP in 184 local residents who had a scheduled regular health checkup, and compared the findings with those for other clinical parameters. Univariate analyses showed that the plasma levels of AM, ANP and BNP were significantly correlated with age. The plasma levels of ANP and BNP were also significantly correlated with systolic blood pressure (SBP) and with pulse pressure (PP), an indicator of the stiffness of the great vessels. Multivariate analyses conducted using a stepwise method revealed that age was a significant, independent variable for the plasma levels of AM, ANP and BNP. In addition, PP was a significant factor for the plasma levels of ANP and BNP, while the plasma AM was significantly associated with body mass index (BMI). Thus, the plasma levels of AM, ANP and BNP all increased in association with aging, and those of ANP and BNP increased in association with PP, suggesting possible relationships between the plasma levels and age-related changes in the cardiovascular system.

When coexpressed with receptor activity-modifying protein (RAMP)2 or -3, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Coexpression of rat (r)CRLR with rRAMP deletion mutants in HEK293T cells revealed that deletion of residues 93^99 from rRAMP2 or residues 58^64 from rRAMP3 significantly inhibits high-affinity [ 125 I]AM binding and AM-evoked cAMP production, despite full cell surface expression of the receptor heterodimer. Apparently, these two seven-residue segments are key determinants of high-affinity agonist binding to rAM receptors and of receptor functionality. Consequently, their deletion yields peptides that are able to serve as negative regulators of AM receptor function. ß

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