Abstract
The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Human CT was purchased from Bachem (Torrance, CA). The human CT receptor cDNA was provided by GlaxoSmithKline
Calcitonin (CT),3 a 32-amino acid peptide secreted by the thyroid gland in response to elevations in blood calcium levels, acts on bone and kidney to maintain calcium homeostasis [1, 2]
Using CT-secretin receptor chimeric analysis, the amino terminus of the CT receptor was identified as a critical region for the action of these small-molecule ligands but with determinants that are distinct from those interacting with the natural ligand, CT
Summary
Materials—Human CT was purchased from Bachem (Torrance, CA). The human CT receptor cDNA was provided by GlaxoSmithKline. Generation of Deletion and Site Mutation Constructs of the CT Receptor—Two deletion and three alanine replacement mutations in the juxtamembranous region of the amino terminus of the human CT receptor were prepared (Fig. 3) These represented deletion of segments 150 –154 (⌬(150 –154)) and 150 –151 (⌬(150 –151)) and substitutions of single amino acid residues Tyr150, Leu151, and Ile153 with an Ala (Y150A, L151A, and I153A). 5 ϫ 105 cells plated on tissue culture plasticware were transfected with 3 g of DNA using a modification of the DEAE-dextran protocol that included 10% dimethyl sulfoxide shock and treatment with 0.1 mM chloroquine diphosphate [18] They were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% FetalClone II. 1.1 Ϯ 0.3 Ͼ1,000 Ͼ1,000 Ͼ1,000 Ͼ1,000 100 Ϯ 27 288 Ϯ 43 Ͼ1,000 12 Ϯ 3 6.6 Ϯ 1.4 4.3 Ϯ 1.1 binding sites/cell ϫ 103
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