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(Journal of Leukocyte Biology. 2002;72:1206-1214.)
© 2002 by Society for Leukocyte Biology

CXCR4 heterogeneity in primary cells: possible role of ubiquitination

Cheryl K. Lapham*, Tatiana Romantseva*, Emmanuel Petricoin{dagger}, Lisa R. King*, Jody Manischewitz*, Marina B. Zaitseva* and Hana Golding*

Divisions of
* Viral Products and
{dagger} Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

Correspondence: Hana Golding, Division of Viral Products, Center for Biologics and Evaluation and Research, Building 29B, Room 4NN04, HFM 454, Bethesda, MD 20892. E-mail: goldingh{at}cber.fda.gov

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.

Key Words: monocytes • lymphocytes • chemokine receptor HIV-1




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