AJP - Gastrointestinal and Liver Physiology, Vol 259, Issue 1 70-G77, Copyright © 1990 by American Physiological Society
Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland
C. R. Marino, J. D. Castle and F. S. Gorelick
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
An antiserum raised against purified rat parotid secretory granule membrane
proteins has been used to identify organelle-specific protein
phosphorylation events following stimulation of intact cells from the rat
parotid gland. After lobules were prelabeled with [32P]orthophosphate and
exposed to secretagogues, phosphoproteins were immunoprecipitated with the
granule membrane protein antiserum, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and visualized by
autoradiography. Parallel studies of stimulated amylase release were
performed. Isoproterenol treatment of parotid lobules resulted in an
increase in the phosphate content of immunoprecipitable 60- and 72-kDa
proteins that correlated with amylase release in a time-dependent manner.
Forskolin addition mimicked these effects, but only the isoproterenol
effects were reversed by propranolol treatment. To confirm the specificity
of the antiserum to the secretory granule membrane fraction, subcellular
isolation techniques were employed following in situ phosphorylation. The
60- and 72-kDa phosphoproteins were immunoprecipitated from both a
particulate fraction and a purified secretory granule fraction.
Furthermore, the extraction properties of both species suggest that they
are integral membrane proteins. These findings support the possibility that
stimulus-regulated secretion may involve phosphorylation of integral
membrane proteins of the exocrine secretory granule.
