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Updated: Sep 28, 2026
A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
Published on: December 2, 2022
Sequencing of a calcitonin receptor-like receptor in salmon Oncorhynchus gorbuscha. Functional studies using the
Elisabeth Pidoux1, Michèle Cressent
1U.349 Institut National de la Santé et de la Recherche Médicale, 6 rue Guy Patin, Centre Viggo Petersen, 75475 Paris Cedex 10, France.
Abstract:
The calcitonin gene-related peptide (CGRP) receptor and adrenomedullin (ADM) receptor are generated by the concomitant expression of a calcitonin receptor-like receptor (CL receptor) and a specific receptor activity-modifying protein (RAMP) in mammals. We have identified the sequence encoding the salmon CL receptor (sCL receptor) and studied its function after co-expression with the human RAMPs in Cos-7 cells. The potential open-reading frame encoded a 465-amino-acid protein which is 72% identical to the human CL receptor and 85.8% identical to the flounder CL receptor. Function was assessed by measuring the cyclic adenosine monophosphate (cAMP) produced by Cos-7 cells transiently transfected with recombinant vectors for the sCL receptor and human RAMP. Co-expression of the CL receptor and RAMP1, formed a CGRP receptor, as in mammals. This CGRP receptor responded to selective analogs as a type 1 CGRP receptor. Cells co-expressing the CL receptor and RAMP2 did not produce increased cAMP in response to human ADM. Cells co-expressing the CL receptor and RAMP3, produced such a response, as in mammals, indicating that the human ADM molecule is not the cause of the previous unresponsiveness. We suggest that the human RAMP2 molecule does not interact with the sCL receptor because of major differences in the sequences of the salmon CL receptor and the mammalian CL receptor. The availability of this receptor must allow to further study their structural basis. This identification of a non-mammalian CL receptor, and characterization of its function, give insight in the evolution of the CL receptor molecule.
Insights
Researchers identified the salmon calcitonin receptor-like receptor (sCL receptor) and studied its function. Co-expression with human receptor activity-modifying proteins (RAMPs) revealed it forms a functional calcitonin gene-related peptide (CGRP) receptor with RAMP1 and an adrenomedullin (ADM) receptor with RAMP3.
Area of Science:
- Biochemistry
- Molecular Biology
- Evolutionary Biology
Background:
- The calcitonin receptor-like receptor (CL receptor) and receptor activity-modifying proteins (RAMPs) form distinct G protein-coupled receptors in mammals.
- These receptors mediate the actions of calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM).
Purpose of the Study:
- To identify and characterize the function of the salmon calcitonin receptor-like receptor (sCL receptor).
- To investigate the interaction of the sCL receptor with human receptor activity-modifying proteins (RAMPs).
Main Methods:
- Sequence identification of the salmon CL receptor (sCL receptor).
- Co-expression of sCL receptor and human RAMPs (RAMP1, RAMP2, RAMP3) in Cos-7 cells.
- Measurement of cyclic adenosine monophosphate (cAMP) production to assess receptor function.
Main Results:
- The sCL receptor shares high sequence identity with mammalian and fish CL receptors.
- Co-expression of sCL receptor and human RAMP1 formed a functional CGRP receptor, responding to selective analogs.
- Co-expression with human RAMP3 formed a functional ADM receptor, while co-expression with RAMP2 did not mediate an ADM response.
Conclusions:
- The sCL receptor can form functional CGRP and ADM receptors with specific human RAMPs.
- Human RAMP2 may not interact with the sCL receptor due to sequence dissimilarities, offering insights into receptor evolution.
- This study provides a non-mammalian CL receptor for further structural and functional studies.
