Multiple growth hormone-binding proteins are expressed on insulin-producing cells.
The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.
Møldrup
A
A
Hagedorn Research Laboratory, Gentofte, Denmark.
Billestrup
N
N
Thorn
N A
NA
Lernmark
A
A
Nielsen
J H
JH
D000345:Affinity Labels; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011986:Receptors, Somatotropin / Q000032:analysis* / Q000302:isolation & purification; D013388:Succinimides
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D011986:Receptors, Somatotropin; D013388:Succinimides; D011092:Polyethylene Glycols; D017830:Octoxynol; C019358:disuccinimidyl suberate