Abstract
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 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, 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.
Footnotes
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This work was supported by the Health Research Council of New Zealand, Lottery Health Commission (New Zealand), Maurice and Phyllis Paykel Trust, and the National Health and Medical Research Council (NHMRC) of Australia (grants 299810 and 436781). P.M.S. is a Principal Research Fellow and A.C., a Senior Research Fellow of the NHMRC.
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ABBREVIATIONS: AMY, amylin receptor phenotype; AM, adrenomedullin; BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl); CGRP, calcitonin gene-related peptide; CL, calcitonin receptor-like receptor; CT(a), insert negative calcitonin receptor isoform; HA, hemagglutinin epitope tag; IMD, intermedin; IMDS, intermedin-short; RAMP, receptor activity modifying protein; WT, wild type; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; ALPHA, amplified luminescent proximity homogenous assay.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received March 17, 2008.
- Accepted July 1, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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