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 (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 alphaCGRP 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.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T2 T30 T31 T32 T33 T34 T35 T36 T37 T38 T39 T3 T40 T41 T42 T43 T44 T45 T46 T47 T48 T49 T4 T50 T51 T52 T53 T54 T55 T56 T57 T58 T59 T5 T60 T61 T62 T63 T64 T6 T7 T8 T9
GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-N GENE-N CHEMICAL GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y
AM RAMP1 alphaCGRP CGRP receptors RAMP RAMP CT(a) receptor RAMP1 S103N W74E N rAmy Calcitonin gene-related peptide (CGRP) receptors CL RAMP1 RAMP3 AM CL RAMP1 adrenomedullin (AM) receptors CL Calcitonin-family receptors RAMP2 AM1 receptor CL RAMP3 AM2 receptor Amylin (Amy) receptors RAMP calcitonin receptor calcitonin receptor-like receptor AMY1 receptor activity-modifying protein AMY2 AMY3 RAMPs G protein-coupled receptor RAMP1 RAMP3 human RAMPs RAMP2 RAMP3 CL RAMPs RAMP1 RAMP2 calcitonin receptor RAMP3 RAMP1 CL RAMP2 CL RAMP3 AM receptors CL RAMP3 RAMP1 adrenomedullin amylin receptor calcitonin gene-related peptide calcitonin receptor CT(a) RAMP1 RAMP3