Abstract
Integrin-associated protein (IAP) is a 50-kDa membrane protein with an amino-terminal immunoglobulin domain and a carboxyl-terminal multiply membrane-spanning region. It is physically and functionally associated with the integrin alpha v beta 3 vitronectin receptor and is involved in the increase in intracellular calcium concentration, which occurs upon cell adhesion to extracellular matrix. Oxidative burst in neutrophils can be induced or inhibited via IAP. Surprisingly, IAP is also expressed on erythrocytes, which have no known integrins. IAP has been shown to be identical to OA3, an ovarian carcinoma antigen. We now show that IAP expression is reduced on Rhnull erythrocytes. The IAP structural gene is mapped to q13.1-2 on human chromosome 3, within a region known to contain a gene encoding the Rh-associated 1D8 antigen. By expression studies on human erythrocytes and IAP transfectants, IAP is shown to be identical to the 1D8 antigen and to CD47, a cell surface protein with broad tissue distribution, reduced in expression on Rhnull erythrocytes. Two CD47 antibodies recognize the immunoglobulin domain of IAP, as does antibody 1D8. These studies suggest the possibility that IAP and the Rh polypeptides may share a pathway for membrane expression on erythrocytes. Furthermore, decreased expression of IAP on Rhnull cells may contribute to the these cells' abnormal cation permeabilities. These studies demonstrate an unexpected link between integrin signal transduction and erythrocyte membrane structure.
Highlights
From the #Division of Infectious Diseases, Department of Medicine, the Wepartmentsof Laboratory Medicine, and the **Department of Surgery, Washington University School of Medicine, St
IAP has a verybroadcellandtissuedistributionandis brane proteinwith an amino-terminal immunoglobulin expressed on all hematopoietic cells, including erythrocytes, domainandacarboxyl-terminalmultiplymembranewhich have no known integrin(s1).From the expression of IAP
Spanning region.It is physically and functionallyasso- on erythrocytes, we reasonedthat IAP might be a blood group ciated with the integrin a,Ps vitronectinreceptor andis antigen
Summary
- b a Results given are maximum inhibition obtained, which in allcases was at a 1:lO dilution of tissue-culture supernatant, the highest concentration tested. The protein was transferred to Immobilon-Ppaper (Millipore, BedfordM, A), blocked with bovine serum albumin, cut into strips, and reacted overnight with the different mAbs as tissue culture supernatants diluted 1:4, followed by detection with peroxidase-conjugatedgoat anti-mouse. Fluorescent Flow Microytometry- Approximately lo cellswere stained and analyzed as described [2] using fluorescein isothiocyanate conjugated rabbit anti-mouse Ig (whole molecule;Sigma) as secondary Ab. Chromosomal Mapping of the IAP Gene- A genomic clone was obtained for use as a probe for fluorescent in situ hybridization. Nick translation with biotin-11-dUTP[20] was used to label 150 ng ofAIAP16,which were hybridized to the chromosomal spreads [21].Subsequently,slides were incubated with fluorescein-conjugated goat anti-avidin D antibody (5pg/ml) and counterstained in the final wash with 4,6-diamidino-2-phenylidone(200 ng/ml) and propidium iodide (200 ng/ml). Cytogenetic banding was done by Giemsa staining using standard methods
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