Modulation of B lymphocyte differentiation by calcitonin gene-related peptide (CGRP). I. Characterization of high-affinity CGRP receptors on murine 70Z/3 cells.
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide found in both peripheral and central neurons and in certain endocrine tissues. Previous studies have identified specific high-affinity CGRP receptors on normal T and B cells and have described modulatory effects of CGRP on freshly isolated lymphocytes. In these studies several lymphocyte cell lines were screened for 125I-CGRP binding. One cell line, the murine 70Z/3 pre-B cell line, was found to exhibit a high level of 125I-CGRP binding and was used for further characterization of the lymphocyte CGRP receptor. Several protease inhibitors were evaluated for their ability to stabilize 125I-CGRP binding to 70Z/3 cells. Bacitracin, a relatively nonspecific protease inhibitor, and chymostatin, a cysteine protease inhibitor, enhanced 125I-CGRP binding, suggesting that 70Z/3 cells express a protease capable of inactivating CGRP. 125I-CGRP binding had a linear dependence on cell concentration with binding being detectable with as few as 200,000 cells/ml and was rapid, with equilibrium binding being reached by 30 min. Saturation binding studies demonstrated that the 70Z/3 CGRP receptor has both low- and high-affinity binding states, with Kds of approximately 0.2 and 3.3 nM, respectively. The density of the low-affinity binding site was approximately 20,000 sites/cell and was unchanged following LPS treatment. In contrast, LPS treatment for 48 hr induced a fourfold increase in the density of the high-affinity site, from 105 to 401 sites/cell. In competition binding studies, only CGRP and the CGRP antagonist CGRP8-37 inhibited 125I-CGRP binding, whereas the unrelated neuropeptides calcitonin, SP, and CRH had no effect. Inhibition of 125I-CGRP binding by rat CGRP was best described by a two-site model, with Kis of 44.1 pM and 0.6 nM. Inhibition of binding by human CGRP and CGRP8-37 was best described by a one-site model with Kis of 3.92 and 6.50 nM, respectively. The 70Z/3 CGRP receptor protein was biochemically characterized by affinity labeling the receptor protein with 125I-CGRP and the covalent cross-linking reagents DSS or BS. SDS-PAGE analysis revealed a major band of 103,000 MW. In addition a high-molecular-weight complex which did not penetrate the gel was also observed. The presence of CGRP receptors on 70Z/3 cells provides further evidence for the immunomodulatory role for CGRP and provides a model system for studying the cellular mechanisms of action of CGRP in modulating lymphocyte differentiation and function.
Department of Microbiology and Immunology, University of Kentucky College of Medicine, Lexington 40536.
J P
JP
McGillis
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Humphreys
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Rangnekar
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Ciallella
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry / Q000502:physiology*; D015740:Calcitonin Gene-Related Peptide / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism
D008070:Lipopolysaccharides; D011480:Protease Inhibitors; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D015740:Calcitonin Gene-Related Peptide