Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.

1 Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. 2 Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC 50 values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+0.20 mM; human=1.90+0.30 mM: CHO cells: rat=0.20+0.06 mM; human=0.19+0.08 mM). 3 In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC 50 values for capsaicin-induced responses were similar in both cell lines (rat=0.35+0.06 mM, human=0.53+0.03 mM). 4 The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC 50 pH 5.49+0.04 and pH 5.78+0.09, respectively). 5 The threshold for heat activation was identical (428C) for rat and human VR1. 6 PPAHV was an agonist at rat VR1 (EC 50 between 3 and 10 mM) but was virtually inactive at the human VR1 (EC 50 410 mM). 7 Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. 8 Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more e ective at inhibiting the noxious heat response of human than of rat VR1.

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is approximately 30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 +/- 0.2 pA/pF in unstimulated cells to 2.9 +/- 0.1 pA/pF for PKA and 3.4 +/- 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump alpha(1)-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

The calcitonin receptor-like receptor (CRLR) can function as either a receptor for calcitonin gene-related peptide (CGRP) or for adrenomedullin (ADM), depending upon the coexpression of a novel family of single transmembrane proteins, which we have called receptor activity modifying proteins or RAMPs. RAMPs 1, 2, and 3 transport CRLR to the plasma membrane with similar efficiencies, however RAMP1 presents CRLR as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor. Characterization of the RAMP2/CRLR and RAMP3/CRLR receptors in HEK293T cells by radioligand binding (125I-ADM as radioligand), functional assay (cAMP measurement), or biochemical analysis (SDS-polyacrylamide gel electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% identity. Chimeric proteins were created with the transmembrane and cytosolic portions of RAMP1 associated with the amino terminus of RAMP2 (RAMP2/1) and vice versa (RAMP1/2). Coexpression of RAMP2/1 with CRLR formed a core glycosylated ADM receptor, whereas the RAMP1/2 chimera generated both core glycosylated and mature forms of CRLR and enabled both ADM and CGRP receptor binding. Hence, the glycosylation state of CRLR appears to correlate with its pharmacology.