Abstract
The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gαs-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gαs and Gαq but also identify a Gαi component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gαs, Gαi, and Gαq/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.
Highlights
Graham Ladds PAGES 21931 AND 21933: There were some errors in Tables 3 and 4 whereby some data values were increased by the addition of 10 units to each data point
Potency, affinity and coupling efficacy values for cAMP production at the CLR co-expressed with each RAMP, stimulated with various agonists measured in HEK-293 cells in the presence and absence of pertussis toxin
The negative logarithm of the agonist concentration required to produce a half-maximal response. b The maximal response to the ligand expressed as a percentage of the maximal cAMP production as determined using 100 M forskolin stimulation in the presence of pertussis toxin treatment. c The negative logarithm of the equilibrium disassociation constant for each ligand generated through use of the operational model of agonism (34). d Log is the coupling efficiency parameter of each ligand
Summary
Potency (pEC50), affinity (pKa) and coupling efficacy (log ) values for cAMP production at the CLR co-expressed with each RAMP, stimulated with various agonists measured in HEK-293 cells in the presence and absence of pertussis toxin. Graham Ladds PAGES 21931 AND 21933: There were some errors in Tables 3 and 4 whereby some data values were increased by the addition of 10 units to each data point. These errors have been corrected and do not affect the results or conclusions of this work.
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