Characterization of amylin binding sites in a human hepatoblastoma cell line.
Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. 125I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (Kd = 0.11 +/- 0.04 nM) and low (Kd = 1.3 +/- 0.4 microM) affinity binding sites for 125I-amylin in HepG2 cells. The dissociation experiments also showed that 125I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing 125I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace 125I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing 125I[Tyr0]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of 125I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8-37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells, and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.
Sheriff
S
S
Department of Surgery, University of Cincinnati Medical Center, OH 45267-0558.
Fischer
J E
JE
Balasubramaniam
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism; D000682:Amyloid; D015740:Calcitonin Gene-Related Peptide; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D005943:Gluconeogenesis / Q000502:physiology; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D008113:Liver Neoplasms / Q000378:metabolism*; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000032:analysis*; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000682:Amyloid; D007457:Iodine Radioisotopes; D058228:Islet Amyloid Polypeptide; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D058287:Receptors, Islet Amyloid Polypeptide; D018000:Receptors, Peptide; D006003:Glycogen; D000262:Adenylyl Cyclases; D015740:Calcitonin Gene-Related Peptide