Characterization of cell surface adenosine 3',5'-monophosphate-binding proteins in Y-1 mouse adrenal tumor cells.
Adrenal cortical cells are known to export cAMP and have binding proteins and cAMP-dependent protein kinase activity associated with their plasma membranes. Because these properties suggest a function for extracellular cAMP, we have undertaken a search for specific cell surface receptors for this cyclic nucleotide. Y-1 mouse adrenal tumor cells actively export cAMP by an energy-dependent process. Analysis of Scatchard plots of the equilibrium binding of [3H]cAMP to these cells indicate the existence of two classes of cAMP binders: one with high affinity (ka = 2.9 X 10(9) M-1) and another with low affinity (ka = 7.0 X 10(7) M-1). The cell surface localization of these binders was established by the sensitivity of both the [3H]cAMP-binding proteins and the [32P]8-N3-cAMP photoaffinity labeled proteins of intact cells to mild trypsin digestion and by the surface distribution of a BSA-O2-monosuccinyl cAMP-gold complex revealed by electron microscopy. Analysis of radioautograms of cell surface cAMP-binding proteins from confluent monolayer tumor cells, photoaffinity labeled with [32P]8-N3-cAMP and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major 32P-labeled protein bands which were indistinguishable from the 49,000 and 55,000 mol wt regulatory subunits of the cytosolic protein kinase isoenzymes of this cell. These observations along with the demonstration of cell surface, cAMP-dependent protein kinase activity in the mouse adrenal tumor cell strongly suggest that these cAMP-binding proteins function as regulatory proteins for cell surface protein kinases.
Wen
S C
SC
Chang
C M
CM
Reitherman
R W
RW
Harding
B W
BW
D000310:Adrenal Gland Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis*; D002478:Cells, Cultured; D003366:Cosyntropin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein; D006046:Gold / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D011494:Protein Kinases / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D008565:Membrane Proteins; D014316:Tritium; D003366:Cosyntropin; D012710:Serum Albumin, Bovine; C017042:8-azidoadenosine-3',5'-monophosphate; C021330:succinyladenosine 3',5'-cyclic monophosphate; D006046:Gold; D000242:Cyclic AMP; D011494:Protein Kinases