The function of conserved cysteine residues in the extracellular domain of human receptor-activity-modifying protein.
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 (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP 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]halphaCGRP 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 halphaCGRP 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.
Steiner
Sarah
S
Departments of Orthopaedic Surgery and Medicine, University of Zurich, Balgrist University Hospital, Forchstrasse 340, 8008 Zurich, Switzerland.
Born
Walter
W
Fischer
Jan A
JA
Muff
Roman
R
D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D058286:Calcitonin Receptor-Like Protein; D000242:Cyclic AMP / Q000096:biosynthesis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis; D011869:Radioligand Assay; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000235:genetics / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins; D000242:Cyclic AMP; D003545:Cysteine