V

V. and protein for was detected in endothelial cells by reverse transcription-PCR and Western blots. When subjected to sequencing by mass spectrometry, the protein was URB602 identified as authentic kindlin-3 and unequivocally distinguished from and or any other known protein. By quantitative real time PCR, the level of kindlin-3 in endothelial cells was 20C50% of that of kindlin-2. Using knockdown approaches, we show that kindlin-3 plays a role in integrin-mediated adhesion of endothelial cells. This function depends upon the integrin and substrate and is distinct from that URB602 of kindlin-2. Formation of tube-like structures in Matrigel also was impaired by kindlin-3 knockdown. Mechanistically, the distinct functions of the kindlins can be traced to differences in their subcellular localization in integrin-containing adhesion structures. Thus, the prevailing view that individual kindlins exert their functions in a cell type-specific manner must now be modified to consider distinct functions of the different URB602 family members within the same cell type. (6). In mammals, there are three kindlin family members, each characterized by a C-terminal FERM domain name bisected by a pleckstrin homology domain name. The FERM domains of kindlins are most closely related Rabbit Polyclonal to HDAC7A (phospho-Ser155) to the FERM domain name of talin, which is also involved in regulation of integrin signaling (7,C11). Kindlins and talin bind to the cytoplasmic tails of integrin subunits via their F3 (PTB) subdomains within their FERM domains. However, the binding sites of talin and kindlins within the cytoplasmic tails do not overlap (5, 12), and the two interactions appear to act cooperatively to optimize integrin activation (12, 13). Hence, cells or mice with decreased kindlin expression levels are unable to properly activate their integrins. Kindlin-1 (UNC-112 related protein 1) is expressed predominantly in epithelial cells; and mutation in the kindlin-1 gene causes Kindler syndrome in humans (14, 15), a rare disease characterized by skin blistering, poikiloderma with frequent intestinal complications. These phenotypes are recapitulated in mice in which the kindlin-1 gene has been inactivated (16). Kindlin-2 (Mig-2) is expressed in most tissues and in many different cell types, and knock-out of kindlin-2 is lethal in mice and zebrafish (13, 17). Mice in which the kindlin-3 gene has been inactivated display defects in platelet (18) and leukocyte (19) responses dependent on integrin activation and the mice die by day 7 postnatally (18) from as yet undefined causes. Recently, kindlin-3 mutations have been identified in humans with a rare syndrome referred to as integrin activation deficiency disease, leukocyte adhesion deficiency III, or LADI (20,C23). The manifestations of the kindlin-3 deficiency include episodic bleeding, susceptibility to frequent infections, and osteopetrosis, which are consequences of an inability to activate 1, 2, and 3 integrins (21, 22). To date, the original publication (4) and all reviews (24, 25) have emphasized that kindlin-3 is restricted to hematopoietic cells and the cellular defects in integrin activation deficiency disease patients support the importance of kindlin-3 in blood cell function. In the present study, using RT2-PCR, Western blotting, and mass spectrometry approaches, we demonstrate for the first time the presence of kindlin-3 in non-hematopoietic cells. Kindlin-3 is an endothelial cell protein, and this point is demonstrated both in cultured endothelial cells from various anatomic origins and mRNA is 50% of that of kindlin-2. Furthermore, kindlin-3 knockdown in endothelial cells results in impaired adhesion to integrin substrates, despite the presence of kindlin-2 in the same cells. These observations suggest different and important roles of both kindlins in integrin signaling in these vascular cells. EXPERIMENTAL PROCEDURES Antibodies and Reagents Rabbit polyclonal antibodies against kindlin-3 were from ProSci Incorporated, mouse monoclonal antibody against EGFP (JL-8) was from Clontech, mouse fluorescein isothiocyanate-labeled monoclonal antibody against human 3 integrin was from BD Biosciences, mouse monoclonal antibody against GAPDH was from Affinity BioReagents, mouse monoclonal antibody against ICAM-1 was from R&D Systems, and rat monoclonal antibody against 1 integrin was from Transduction Laboratories. Mouse monoclonal antibody against kindlin-2 was provided.