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Characterization of Insulin-Like Growth Factor-Binding Proteins from Sheep Thyroid Cells^*

Department of Pediatrics, Stanford University Medical Center Stanford, California 94305 The Department of Medicine, University of California San Diego La Jolla, California 92093–0613

Address all correspondence and requests for reprints to: Dr. Laura K. Bachrach, Department of Pediatrics, Room S322, Stanford University Medical Center, Stanford, California 94305.

Abstract

The insulin-like growth factors (IGF_s) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when crosslinked to [¹²⁵I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40–44 and 48–52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [¹²⁵I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGFstimulated binding proteins to 36–40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36–38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are nonglycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins antigenically related to BP-53.

Footnotes

^* This work was supported by NIDDK Grant 1-R29-DK-40317–01.

Received May 30, 1989.

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