Identification of R-GRAMP, a membrane glycoprotein of regulated secretory granules in primate cells.
Investigation of membrane assembly and traffic in the regulated secretory pathway may be facilitated by identification of membrane components that are unique to regulated secretory granules. To identify such markers, we isolated integral membrane proteins by Triton X-114 extraction from well-differentiated monolayers of an exocrine cell line, the goblet cell subclone (18N2) of the human colon carcinoma cell line HT29, and used the extracts as immunogens to produce monoclonal antibodies (mAbs). Immunofluorescence microscopy of HT29 goblet cell monolayers identified one mAb (MG-1) that labeled a component of mucin granule membranes. Immunofluorescence of frozen semithin sections of normal intestine, and various other human and monkey tissues, showed that this antigen is present in regulated secretory granule membranes of primate exocrine cells, endocrine cells, and tissue granulocytes. EM immunogold labeling of goblet cells, enteroendocrine cells and eosinophils confirmed that the antigen is associated with secretory granule membranes and not with plasma membranes. The antigen was identified by SDS-PAGE autoradiography of immunoprecipitates from HT29 goblet cells metabolically labeled with [35S]methionine and [35S]cysteine or [3H]glucosamine, as a glycoprotein with an apparent molecular mass ranging from 23 to 37 kDa. Digestion of immunoprecipitates with N-glycosidase F reduced the apparent mass to 16 to 19 kDa. This small, highly-glycosylated protein was named "R-GRAMP" (for regulated granule-associated membrane protein) to reflect its wide distribution in secretory granule membranes of regulated exocrine, endocrine and granulocytic cell types. This distribution suggests that it may play a common functional role in regulated secretion.
Reinhart
F D
FD
GI Cell Biology Research Laboratory, Children's Hospital, Boston, MA 02115.
Phillips
T E
TE
Trahair
J F
JF
Neutra
M R
MR
D001078:APUD Cells / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000737:chemistry*; D004804:Eosinophils / Q000737:chemistry; D005088:Exocrine Glands / Q000737:chemistry / Q000166:cytology; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000737:chemistry / Q000166:cytology; D008562:Membrane Glycoproteins / Q000032:analysis*; D009474:Neurons / Q000737:chemistry; D011323:Primates / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins