Characterization of/3pat-3 Heterodimers, a Family of Essential Integrin Receptors in C. elegans

Members of the integrin family of cell surface receptors have been shown to mediate a diverse range of cellular functions that require cell-cell or cell-extracellular matrix interactions. We have initiated the characterization of integrin receptors from the nematode Caenorhabditis elegans, an organism in which genetics can be used to study integrin function with single cell resolution. Here we report the cloning of an integrin/3 subunit from C. elegans which is shown to rescue the embryonic lethal mutation pat3(rh54) and is thus named/3pat-3. Analysis of the deduced amino acid sequence revealed that/3pat-3 is more similar to Drosophila integrin/3PS and to vertebrate integrin/31 than to other integrin/3 subunits. Regions of highest homology are in the RGD-binding region and in the cytoplasmic domain. In addition, the 56 cysteines present in the majority of integrin/3 subunits are conserved. A major transcript of ~3 kilobase pairs was detected by RNA blot analysis. Immunoblot analysis using a polyclonal antiserum against the cytoplasmic domain showed that/3pat-3 migrates in SDS-PAGE with apparent M~ of 109 k and 120 k under nonreducing and reducing conditions, respectively. At least nine protein bands with relative molecular weights in the range observed for known integrin a subunits coprecipitate with/3pat-3, and at least three of these bands migrate in SDS-PAGE with increased mobility when reduced. This behavior has been observed for a majority of integrin tx subunits. Immunoprecipitations of/3pat-3 from developmentally staged populations of C. elegans showed that the expression of several of these bands changes during development. The monoclonal antibody MH25, which has been postulated to recognize the transmembrane component of the muscle dense body structure (Francis, G. R., and R. H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:1532-1549), was shown to recognize/3pat-3. Finally, immunocytochemical analysis revealed that/3pat-3 is expressed in the embryo and in many cell types postembryonically, including muscle, somatic gonad, and coelomocytes, suggesting multiple roles for integrin heterodimers containing this/3 subunit in the developing animal. T HE interactions of cells with their environments are critical for the development and maintenance of tissues in multiceUular organisms. Members of one family of cell surface receptors, the integrins, have been shown to mediate biological processes requiring cell-cell or cellextracellular matrix adhesion such as cell attachment, cell migration, and neurite outgrowth (for review see Hynes, 1992). Each receptor is a heterodimer composed of noncovalently associated ct and 13 subunits. To date, fourteen subunits and eight/3 subunits have been identified in verPlease address all correspondence to L. E Reichardt, Howard Hughes Medical Institute, Room U-426, University of California, San Francisco, Box 0724, Third and Parnassus Avenues, San Francisco, CA 94143-0724. Tel.: (415) 476-3976. Fax: (415) 566-4969. The current address of S. N. Gettner is Department of Oral Craniofacial Biological Sciences, Dental School, University of Maryland, Baltimore, MD 21201. tebrates. Ligands of integrin receptors include extracellular matrix proteins, such as laminin, fibronectin, and several collagens, as well as cell surface proteins, such as VCAM-1 and the three ICAMs. In vitro studies and immunocytochemical analyses have suggested several roles for integrins in developmental processes. However, few experiments have addressed integrin function during development in vivo. In chick, injection of an anti-/31 antibody has been shown to interfere with neural crest cell migration (Bronner-Fraser, 1986), and anti-sense experiments have suggested that/31 integrins are required for neuroblast migration in the optic tectum (Galileo et al., 1992). In Pleurodeles embryos, injection of anti-/31 antibodies was found to inhibit gastrulation (Darribere et al., 1988). To date, the analysis of the Drosophila melanogaster integrin /3PS subunit mutant lethal(1)myospheroid has been the most comprehensive study of integrin function during develop© The Rockefeller University Press, 0021-9525/95/05/i 127/15 $2.00 The Journal of Cell Biology, Volume 129, Number 4, May 1995 1127-1141 1127 on F ebuary 3, 2013 jcb.rress.org D ow nladed fom Published May 15, 1995