Identification of N-Terminal Receptor Activity-Modifying Protein Residues Important for Calcitonin Gene-Related Peptide, Adrenomedullin, and Amylin Receptor Function
Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM 1 receptor) or CL/RAMP3 (A...
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Published in | Molecular pharmacology Vol. 74; no. 4; pp. 1059 - 1071 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Pharmacology and Experimental Therapeutics
01.10.2008
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Subjects | |
Online Access | Get full text |
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Summary: | Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying
protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors
are CL/RAMP2 (AM 1 receptor) or CL/RAMP3 (AM 2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY 1 , AMY 2 , and AMY 3 , respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences
between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs
revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in
RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis,
we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were
transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT (a) ]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3
was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to αCGRP potency at
CGRP receptors. Mutant RAMP/CT (a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy
potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based
receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3
as a key determinant of AM pharmacology. |
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ISSN: | 0026-895X 1521-0111 |
DOI: | 10.1124/mol.108.047142 |