The function of extracellular cysteines in the human adrenomedullin receptor.
When co-expressed with receptor activity-modifying protein (RAMP) 2, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2). In the present study, we examined the function of the cysteine (C) residues in the extracellular loops of human (h)CRLR (C212, C225 and C282) and in the extracellular domain of hRAMP2 (C68, C84, C99 and C131). Using site-directed mutagenesis, the cysteine residues were substituted, one at a time, with alanine (A). Co-expression in HEK293 cells of hRAMP2 with the hCRLR C212A or C282A mutant significantly reduced the 50% of effective concentration (EC50) for AM-evoked cyclic adenosine monophosphate (cAMP) production, despite full cell surface expression of the receptor heterodimer. Co-expression of the C225A mutant had no effect on [125I]AM binding or receptor signaling. These results suggest that the cysteine residues in the first (C212) and the second (C282) extracellular loops form a disulfide bond that is important for stabilizing the receptor in the correct conformation for ligand binding and activation. Cells expressing hCRLR with an hRAMP2 mutant (C68A, C84A, C99A or C131A) showed no specific AM binding or AM-stimulated cAMP accumulation. Though abundant in the intracellular compartment, these receptors were not detected at the cell surface, suggesting that all four cysteine residues are essential for efficient transport to the plasma membrane. Cysteine residues in the extracellular loops of hCRLR and in the extracellular domain of hRAMP2 thus appear to play distinct roles in the cell surface expression and function of the receptor heterodimer.
Kuwasako
Kenji
K
First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. kuwasako@fc.miyazaki-med.ac.jp
Kitamura
Kazuo
K
Uemura
Tomohiko
T
Nagoshi
Yasuko
Y
Kato
Johji
J
Eto
Tanenao
T
D058286:Calcitonin Receptor-Like Protein; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; C545503:RAMP2 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018000:Receptors, Peptide; D000242:Cyclic AMP; D003545:Cysteine