Search

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys27 in myc-tagged human (h) RAMP1 was deleted (hRAMP1Δ1), and Cys40, Cys57, Cys72, Cys82 and Cys104 were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Δ1 or -C82A, cell surface expression, [125I]hαCGRP binding and cAMP formation in response to hαCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24±7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [125I]hαCGRP binding of hCLR/myc-hRAMP1-C72A was 13±3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by hαCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14±1% and 33±2% of that of the hCLR/myc-hRAMP1 with comparable EC50. But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys27 or the substitution of Cys82 by Ala in hRAMP1, but it was impaired by the substitution of Cys40, Cys57, Cys72 and Cys104 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.

In Xenopus oocytes with an endogenous calcitonin gene-related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333-339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) alphaCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist alphaCGRP(8-37). Co-expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to h alphaCGRP by 47% (P0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22-52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.

The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn(60), Asn(112) and Asn(117) are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [(125)I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn(117), and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn(117) by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.

Differential glycosylation of human and rat (r) calcitonin (CT) receptor-like receptors (CRLR) as a result of interactions with accessory receptor activity-modifying proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [(125)I]CGRP or [(125)I]ADM. In conclusion, [(125)I]CGRP/rCRLR/RAMP1 and [(125)I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.

The calcitonin (CT)-like (CL) receptor is a CT gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor when co-expressed with receptor-activity-modifying proteins (RAMP) 1 or 2, respectively. The CL receptor shows 57% overall sequence identity with the CT receptor, but the homology is much lower in the extreme N-terminus. An N-terminal deletion mutant of the human (h) CL receptor (Delta18-hCL) and a chimeric receptor consisting of the N-terminal amino acids of the porcine (p) CT receptor fused to the Delta18-hCL receptor (pCT-hCL) were therefore analyzed. The Delta18-hCL receptor function was abolished when co-expressed with RAMP1 or -2. The pCT-hCL receptor was a fully functional CGRP receptor when co-expressed with RAMP1, but the RAMP2-dependent AM receptor function was impaired. Limited sequence similarities in the N-terminus of the pCT and the hCL receptors rescue CGRP but not AM receptor binding and signalling.

Unlike in other endocrine systems calcium inhibits parathyroid hormone (PTH) secretion and this inhibition is paralleled by a rise of cytosolic calcium concentration ([Ca]i). Because of evidence that diglyceride levels and protein kinase C activity are also decreased by high extracellular calcium we have investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, on [Ca]i and PTH secretion using dispersed bovine parathyroid cells. At 1.5 mM medium calcium TPA enhanced PTH secretion and caused reduction of [Ca]i from 639 +/- 36 nM (SE) to 335 +/- 21 nM (P less than 0.001); at 0.5 mM calcium TPA was ineffective. Moreover, TPA suppressed the rise of [Ca]i evoked by high extracellular calcium. Thus TPA presumably stimulates PTH secretion via activation of protein kinase C, and the lowering of [Ca]i may TPA presumably stimulates PTH secretion via activation of protein kinase C, and the lowering of [Ca]i may be a secondary event related to diglyceride availability.