Abstract
When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91-94, 96-100, or 101-103 blocked [125I]CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78-80 or 88-90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91-103 one at a time had little effect on CGRP-induced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101-103 when co-transfected with hCRLR, and expression of a L94A/D101-103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.
Highlights
Ring structures comprised of six amino acids linked by a disulfide bridge and an amidated C terminus that are required for biological activity [3]
To assess which regions of the calcitonin receptor-like receptor (CRLR)/RAMP1 heterodimer are involved in binding hCGRP or hAM and mediating cAMP accumulation, we constructed a group of hRAMP1 mutants with various deletions in their extracellular N-terminal domains (Fig. 1)
When receptor activity-modifying protein (RAMP) chimeras were co-transfected into cells together with CRLR, their binding characteristics and functionality revealed that the determinants of calcitonin gene-related peptide (CGRP) and AM selectivity reside in the extracellular domains of RAMP
Summary
Reagents—[125I]h␣CGRP (specific activity 2 Ci/pmol) was produced in our laboratory using a modification of a method described previously [2]. To evaluate cell surface expression, cells were harvested following transient transfection, washed twice with PBS, resuspended in ice-cold FACS buffer [9], and incubated with anti-HA-FITC antibody (1:50 dilution) for 60 min at 4 °C in the dark. MRNA Expression Measured by Real-time Quantitative PCR—Total RNA was extracted from transfected HEK-293 or HEK-293T cells using Total RNA isolation reagent (Invitrogen) and reverse-transcribed using SuperScript reverse transcriptase (Invitrogen), yielding the respective cDNAs. The expression of mRNAs encoding hCRLR, the three hRAMP isoforms, and hAM was assessed using real-time quantitative PCR (Prism 7700 Sequence Detector, Applied Biosystems, Foster City, CA) as described previously [17, 18].
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