Human Receptor Activity-modifying Protein Research Articles

Receptor activity modifying protein‐1 (RAMP1) overexpression selectively enhances calcitonin gene‐related peptide‐induced vasodilation

Calcitonin gene‐related peptide (CGRP), a potent vasodilator, is released during migraine and mediates vasodilation in response to trigeminal nerve activation. CGRP receptors consist of a calcitonin‐like receptor (CLR) and a receptor activity modifying protein‐1 (RAMP1). The aim of this study was to test whether RAMP1 overexpression enhances vasodilation to CGRP. Transgenic mice overexpressing human RAMP1 in most tissues, including blood vessels, were generated using a RAMP1 transgene under control of the widely expressed adenoviral EIIa promoter. In carotid artery in vitro, maximum relaxation to CGRP was increased ∼ 2‐fold in transgenic vs littermate controls, an effect that was inhibited by the CGRP receptor antagonist, CGRP (8–37) (P<0.05). Responses to adrenomedullin (which activates CLR coupled with RAMP2) were similar in both groups (P>0.05). In cerebral circulation, responses to CGRP were selectively increased ∼ 2‐fold in cerebral arterioles in vivo, and ∼ 70% in the basilar artery in vitro in transgenic vs control mice (P<0.05). These data indicate that RAMP1 overexpression selectively enhances vasodilation to CGRP, suggesting responses to CGRP are normally RAMP1‐limited. RAMP1 may represent a therapeutic target to inhibit increased responses to CGRP during migraine, or increase vasodilator responses and CGRP‐mediated signaling in conditions such as ischemia or vasospasm.

Post-endocytic Sorting of Calcitonin Receptor-like Receptor and Receptor Activity-modifying Protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Functions of the Cytoplasmic Tails of the Human Receptor Activity-modifying Protein Components of Calcitonin Gene-related Peptide and Adrenomedullin Receptors

Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in (125)I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.

Identification of the Human Receptor Activity-modifying Protein 1 Domains Responsible for Agonist Binding Specificity

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.

Sequencing of a calcitonin receptor-like receptor in salmon Oncorhynchusgorbuscha. Functional studies using the human receptor activity-modifying proteins

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.