Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes.
Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide shown to be cosecreted with insulin from the pancreatic beta-cells. We have investigated the existence and characteristics of IAPP binding sites in the rat. Specific binding sites for [125I]IAPP were found to be highest in the lung followed by the stomach fundus, spleen, brain stem, hypothalamus, and the liver, respectively. The interaction of [125I]IAPP with its binding site was rapid and temperature dependent, displaying optimum binding at 4 C. This may be explained by the rapid degradation of the label observed at 22 C and 37 C, as determined by fast protein liquid chromatography analysis, and also degradation of the receptor at 37 C. Binding of [125I]IAPP was rapidly dissociated by the addition of 200 nM unlabeled peptide. The presence of nonmetabolizable GTP-gamma-S (0.5 microM) reduced binding, thus suggesting the coupling of the binding site to a G protein. Rat IAPP displaced [125I]IAPP displaying an IC50 of 5.75 x 10(-9) M (mean, n = 4). Displacement was also seen with human IAPP (IC50 = 5.53 x 10(-8) M), human alpha-calcitonin gene-related peptide (CGRP) (IC50 = 3.8 x 10(-8) M), rat alpha-CGRP (IC50 = 9.0 x 10(-7) M), and rat beta-CGRP (IC50 = 5.53 x 10(-8) M); suggesting an IAPP-specific binding site. Scatchard plots for rat IAPP binding in the lung gave a dissociation constant of 10.4 +/- 2.63 nM (mean +/- SE, n = 4) and maximal binding of 3.1 +/- 0.97 pmol/mg (mean +/- SE, n = 4), displaying a single class of binding site. Chemical cross-linking analysis showed binding of IAPP to sites of Mr 67,000, 64,000, and 38,000. These findings suggest that specific IAPP binding sites exist which differ from the CGRP receptors in rat tissues. This indicates a possible novel autocrine/paracrine role for IAPP.
Bhogal
R
R
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Smith
D M
DM
Bloom
S R
SR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000682:Amyloid / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D013045:Species Specificity
D000682:Amyloid; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D011956:Receptors, Cell Surface; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate