MOLECULAR-IDENTIFICATION OF BINDING-SITES FOR CALCITONIN-GENE-RELATED PEPTIDE (CGRP) AND ISLET AMYLOID POLYPEPTIDE (IAPP) IN MAMMALIAN LUNG - SPECIES VARIATION AND BINDING OF TRUNCATED CGRP AND IAPP

We have investigated the binding of rat [I-125] islet amyloid polypeptide (IAPP) and [I-125]calcitonin gene-related peptide (CGRP) to lung membranes from the rat, rabbit, and bull and have characterized the MOI Wt (M(r)) of the binding site for each ligand by chemical cross-linking. Results imply the existence of three distinct types of binding site demonstrated by both [I-125]CGRP and [I-125]IAPP in each of the three species investigated. These were differentiated by the relative potencies of displacement by rat CGRP, human CGRP-(8-37), rat IAPP, and the rat IAPP fragments IAPP-(8-37), IAPP-(12-37), IAPP-(25-37), and IAPP-(28-37). High affinity binding sites were identified for [I-125]CGRP [rat K(i), 0.119 +/- 0.027 nm (n = 6); rabbit K(i), 0.944 +/- 0.075 nm (n = 6); bull K(i), 0.20 +/- 0.031 nm (n = 6)], and CGRP-(8-37) was found to displace [I-125]CGRP in all species [rat K(i), 6.63 +/- 0.91 nm (n = 6); rabbit K(i), 22.70 +/- 3.79 nm (n = 6); bovine K(i), 26.9 +/- 0.21 nm (n = 3)]. Compared to CGRP-(8-37), displacement by IAPP also showed varying affinities that were similar to that of CGRP-(8-37) (rat), lower than that of CGRP-(8-37) (rabbit), or higher than that of CGRP-(8-37) (bull). Truncation of IAPP caused large parallel decreases in its affinity for [I-125]CGRP in the rabbit and bull by the loss of residues 1-8 (rabbit) and 1-12 (bull), but was not as pronounced in the rat. [I-125]IAPP demonstrated high affinity binding in each species [rat K(i), 5.86 +/- 0.86 nm (n = 6); rabbit K(i), 18.72 +/- 2.90 nm (n = 6); bull K(i), 1.97 +/- 0.40 nm (n = 6)]. Truncation of IAPP caused a reduction of its affinity for [I-125]IAPP in all species by the loss of residues 1-28. Chemical cross-linking analysis indicated binding of both ligands to sites of 64,000 M(r) in the rat and 50,500 and 51,000 M(r) in the rabbit and bull, respectively. In addition, [I-125]IAPP bound to to a site of 100,000 M(r) in the rat. [I-125]CGRP and [I-125]IAPP binding were reduced in the presence of guanosine 5-o-(3-Thiotriphosphate) in all species, indicating an association with G-proteins. This study implies the existence of CGRP/IAPP-binding sites in the lungs of these species that show varying and complex patterns of displacement by CGRP, IAPP, and their fragments.