title: Complementary methods provide evidence for the expression of CXCR7 on human B cells.
abstract: PTMs of extracellular domains of membrane proteins can influence antibody binding and give rise to ambivalent results. Best proof of protein expression is the use of complementary methods to provide unequivocal evidence. CXCR7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL12 and CXCL11. The expression of CXCR7 on nonhematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was recently challenged. To solve the dissent, we thoroughly analyzed the expression of CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. Examination of CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human B cells. Moreover, real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally, two CXCR7-specific peptides were identified by MS in immunoprecipitates from primary human B cells. Thus, we present a strategy based on combined proteomic and functional approaches that can be used to solve dissents on protein expression, i.e. demonstrating the expression of CXCR7 on human leukocytes.
authors.affiliation: Insitute for Research in Biomedicine, Bellinzona, Switzerland.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Humpert
authors.forename: Manuel
authors.initials: M
authors.lastname: Tzouros
authors.forename: Sylvia
authors.initials: S
authors.lastname: Thelen
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bignon
authors.forename: Angélique
authors.initials: A
authors.lastname: Levoye
authors.forename: Fernando
authors.initials: F
authors.lastname: Arenzana-Seisdedos
authors.forename: Karl
authors.initials: K
authors.lastname: Balabanian
authors.forename: Françoise
authors.initials: F
authors.lastname: Bachelerie
authors.forename: Hanno
authors.initials: H
authors.lastname: Langen
authors.forename: Marcus
authors.initials: M
authors.lastname: Thelen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001402:B-Lymphocytes / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D014066:Palatine Tonsil / Q000166:cytology; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D054387:Receptors, CXCR / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C501896:ACKR3 protein, human; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D054387:Receptors, CXCR