Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.
Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.
Miettinen
H M
HM
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Matter
K
K
Hunziker
W
W
Rose
J K
JK
Mellman
I
I
D000199:Actins; D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009154:Mutation; D011961:Receptors, Fc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D000199:Actins; D000936:Antigen-Antibody Complex; D011961:Receptors, Fc; D014443:Tyrosine