title: CXCR4 heterogeneity in primary cells: possible role of ubiquitination.
abstract: The chemokine receptor CXCR4 is a primary coreceptor for the HIV-1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T-lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, antiubiquitin antibodies reacted with the 62-kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101-kDa CXCR4 isoform in immunoblots by three- to sevenfold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV-1 envelope-expressing cells. Similar increases in the intensity of the 101-kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.
authors.affiliation: Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: Cheryl K
authors.initials: CK
authors.lastname: Lapham
authors.forename: Tatiana
authors.initials: T
authors.lastname: Romantseva
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Petricoin
authors.forename: Lisa R
authors.initials: LR
authors.lastname: King
authors.forename: Jody
authors.initials: J
authors.lastname: Manischewitz
authors.forename: Marina B
authors.initials: MB
authors.lastname: Zaitseva
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018740:Genetic Heterogeneity; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000737:chemistry; D008264:Macrophages / Q000737:chemistry; D008970:Molecular Weight; D009000:Monocytes / Q000737:chemistry; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021381:Protein Transport; D019718:Receptors, CXCR4 / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D014452:Ubiquitins / Q000302:isolation & purification
chemical_list: D020033:Protein Isoforms; D019718:Receptors, CXCR4; D014452:Ubiquitins; C067713:lactacystin; D000111:Acetylcysteine