The seven amino acids of human RAMP2 (86) and RAMP3 (59) are critical for agonist binding to human adrenomedullin receptors.
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.
Kuwasako
K
K
First Department of Internal Medicine, Miyazaki Medical College, Miyazaki 889-1692, Japan. kuwasako@fc.miyazaki-med.ac.jp
Kitamura
K
K
Ito
K
K
Uemura
T
T
Yanagita
Y
Y
Kato
J
J
Sakata
T
T
Eto
T
T
D053607:Adrenomedullin; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D000242:Cyclic AMP / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018015:Receptors, Calcitonin Gene-Related Peptide; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D053607:Adrenomedullin; D000242:Cyclic AMP