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The function of conserved cysteine residues in the extracellular domain of human receptor‐activity‐modifying protein 1
Author(s) -
Steiner Sarah,
Born Walter,
Fischer Jan A.,
Muff Roman
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01250-x
Subject(s) - extracellular , transmembrane domain , cysteine , receptor , chemistry , transmembrane protein , calcitonin gene related peptide , microbiology and biotechnology , peptide , calcitonin receptor , function (biology) , biochemistry , biology , neuropeptide , enzyme
The receptor‐activity‐modifying protein (RAMP) 1 is a single‐transmembrane‐domain protein associated with the calcitonin‐like receptor (CLR) to reveal a calcitonin gene‐related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys 27 in myc‐tagged human (h) RAMP1 was deleted (hRAMP1Δ1), and Cys 40 , Cys 57 , Cys 72 , Cys 82 and Cys 104 were each replaced by Ala. In COS‐7 cells expressing hCLR/myc‐hRAMP1Δ1 or ‐C82A, cell surface expression, [ 125 I]hαCGRP binding and cAMP formation in response to hαCGRP were similar to those of hCLR/myc‐hRAMP1. Cell surface expression of myc‐hRAMP1‐C72A was reduced to 24±7% of myc‐hRAMP1, and that of ‐C40A, ‐C57A and ‐C104A was below 10%. [ 125 I]hαCGRP binding of hCLR/myc‐hRAMP1‐C72A was 13±3% of hCLR/myc‐hRAMP1 and it was undetectable in hCLR/myc‐hRAMP1‐C40A‐, ‐C57A‐ and ‐C104A‐expressing cells. Maximal cAMP stimulation by hαCGRP in hCLR/myc‐hRAMP1‐C40A‐ and ‐C72A‐expressing cells was 14±1% and 33±2% of that of the hCLR/myc‐hRAMP1 with comparable EC 50 . But cAMP stimulation was abolished in cells expressing hCLR/myc‐hRAMP1‐C57A and ‐C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys 27 or the substitution of Cys 82 by Ala in hRAMP1, but it was impaired by the substitution of Cys 40 , Cys 57 , Cys 72 and Cys 104 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.