Cloning and cellular localization of the rat mast cell 78-kDa protein phosphorylated in response to the mast cell "stabilizer" cromolyn.
Disodium cromoglycate (cromolyn) inhibits mast cell secretion, but its mechanism has not been elucidated. One possibility is the phosphorylation of a 78-kDa mast cell protein, two fragments of which are homologous to moesin, a member of the ezrin, radixin, moesin family. These proteins appear to be involved in signal transduction by regulating functional associations between the cell surface and the cytoskeleton. Moesin cDNA was cloned from rat basophil leukemia cells, which are similar to mucosal mast cells, and polyclonal antiserum was prepared against recombinant moesin expressed in Escherichia coli. Moesin phosphorylated in mast cells treated with cromolyn shifted from the soluble to the precipitable fraction and associated with Sepharose-linked beta-actin. Recombinant moesin also associated with Sepharose-linked beta-actin, and so did purified RBL moesin, but only if the latter was first denatured. Moesin thus appears to have actin binding sites that are not exposed under normal conditions but may become available by in vivo phosphorylation or by denaturation. Immunocytochemistry using confocal microscopy showed moesin to be primarily localized on the inner aspect of the plasma membrane and around secretory granules. Double immunocytochemistry for moesin and actin colocalized them in most areas. Ultracryoimmunoelectron microscopy to preserve the antigenicity of moesin identified the protein close to the plasma and secretory granule membranes. Cromolyn appeared to induce clustering of moesin around secretory granules. It is hypothesized that conformational changes of moesin, regulated by phosphorylation/dephosphorylation, may lead to positional rearrangements with respect to the membrane/cytoskeleton that could possibly regulate mast cell secretion.
Theoharides
T C
TC
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA, USA. Theoharis.Theoharides@tufts.edu
Wang
L
L
Pang
X
X
Letourneau
R
R
Culm
K E
KE
Basu
S
S
Wang
Y
Y
Correia
I
I
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004205:Cromolyn Sodium / Q000494:pharmacology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D007106:Immune Sera; D007150:Immunohistochemistry; D008297:Male; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000199:Actins; D018076:DNA, Complementary; D007106:Immune Sera; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C071058:moesin; D004205:Cromolyn Sodium