Abstract
When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptor-like receptor (CRLR) can function as a calcitonin gene-related 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 Trp(86) (Ala(87)), Met(88), Ile(89), Ser(90), Arg(91), or Pro(92) 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.
Highlights
CGRP1 and AM belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators [1, 2]
We found that AM but not CGRP increased the cAMP content of untransfected cells slightly; maximal cAMP levels reached ϳ2.5-fold over baseline, which was consistent with the finding that intact HEK293 cells endogenously express human RAMP2 (hRAMP2)
We have shown that seven amino acids located between three conserved residues in the extracellular N-terminal domains of hRAMP2 (86 –92) and hRAMP3 (59 – 65) are essential for high affinity agonist binding to hAM receptors as well as for the functionality of the receptor
Summary
CGRP1 and AM belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators [1, 2] 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). Our results indicate that seven-residue segments situated between three residues conserved in both hRAMP2 and hRAMP3 (amino acids 86 –92 and 59 – 65, respectively) are key determinants of high affinity agonist binding to the hAM receptors
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