The gene product of the gp78/AMFR ubiquitin E3 ligase cDNA is selectively recognized by the 3F3A antibody within a subdomain of the endoplasmic reticulum.
The receptor for the autocrine motility factor/phosphoglucose isomerase cytokine (gp78 or AMFR) has been extensively characterized using the 3F3A monoclonal antibody. Cloning of AMFR identified a seven-transmembrane domain G-protein-coupled receptor ubiquitin E3 ligase whose identity as AMFR was based on prior expression cloning with the 3F3A mAb that generated a truncated sequence. We show here that the gp78/AMFR gene product is indeed recognized by the 3F3A mAb. The FLAG-taggedAMFR immunoprecipitated with an anti-FLAG antibody was recognized by the 3F3A mAb in Western blot analysis and cells transfected with AMFR exhibit increased labeling with the 3F3A mAb. The 3F3A mAb does not however recognize higher molecular weight isoforms of AMFR. 3F3A labeling colocalizes with tagged AMFR in a peripheral ER network but does not recognize FLAG- or GFP-tagged AMFR localized to a perinuclear ER domain that likely corresponds to misfolded forms of the protein retained in the ER. These data indicate that 3F3A antibody binding is highly specific for a subpopulation of AMFR localized to an ER subdomain. Coexpression of AMFR-GFP and a lumenal ER-targeted RFP presented extensive colocalization in living cells andAMFR-GFP is concentrated in a basal ER network morphologically similar to that labeled by the 3F3A mAb in fixed cells. The3F3A anti-AMFR mAb therefore selectively recognizes a subpopulation of expressed AMFR localized to a subdomain of the ER.
Département de pathologie et biologie cellulaire, Université de Montréal, Que., Canada.
Marilyn
M
Registre
Jacky G
JG
Goetz
Pascal
P
St Pierre
Hao
H
Pang
Monique
M
Lagacé
Michel
M
Bouvier
Phuong U
PU
Le
Ivan R
IR
Nabi
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism*; D007668:Kidney / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases