Abstract
Integrins of the beta7 subfamily, alpha4 beta7 and alphaE beta7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The beta subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin beta7 cytoplasmic tail and is homologous to mouse EED and Drosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of alpha4 and alphaE but not to those of integrin beta1, beta2, and alphaL subunits. Association of WAIT-1 and beta7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the beta7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.
Highlights
Integrins of the 7 subfamily, ␣47 and ␣E7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites
Molecular Cloning of WAIT-1, a Human WD Repeat Protein Interacting with 7-Integrin Cytoplasmic Domains—The complete 52-amino acid cytoplasmic tail of the human integrin 7 subunit was fused in frame with the lexA DNA binding domain and used as a bait protein for yeast interaction trap screening of a Jurkat cDNA library
Surface receptors of the integrin family are engaged in regulated cell adhesion and ligand-induced signal transduction events with the cytoplasmic tails of ␣ and  subunits being of critical importance
Summary
Antibodies and Cell Lines—The rat-anti-murine integrin 7 antibody M301 was kindly provided by Dr P. All cytoplasmic tail constructs were verified by double-stranded nucleotide sequencing and subcloned into plex202 to generate fusion proteins with the lexA DNA binding portion. Following addition of the peptide MDYKDDDDKGG to the N terminus of WAIT-1, the 5Ј cDNA fragment isolated by nested RACE-PCR was used to generate the full-length protein. GST fused to amino acids 463–511 of the murine zinc finger transcription factor Egr-1 was used as control Both constructs were verified by doublestranded nucleotide sequencing. Association of WAIT-1 and 7-Integrin in Vivo—cDNA clones encoding the full-length murine ␣4 or 7 subunits were subcloned into the expression vectors pRK5 (Pharmingen, Hamburg, Germany) or pSR␣ [29], respectively. Full-length WAIT-1 was fused to the C terminus of domains 2 and 3 of the human IgG1 constant region and subcloned into the pCDM7 vector generating the WAIT-Fc fusion protein. The Northern blot containing mRNA of various human tissues was purchased from CLONTECH. cDNA probes spanning the coding region of wait-1 or -actin (CLONTECH) were labeled with [␣-32P]dCTP (Amersham) using the high prime kit (Boehringer, Mannheim, Germany) and used for hybridization according to the manufacturer’s instructions (CLONTECH)
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