Re-epithelialization of the alveolar basement membrane is a crucial aspect of lung repair after injury, a process that requires the proliferation of type II cells and their differentiation into type I cells. Despite the importance of this process, and significant investigative efforts, only limited information is available regarding the signals that mediated this process. Since, in other systems receptor tyrosine kinases play an important role in cell growth and differentiation, we hypothesized that, (1) As yet unidentified epithelial cell-specific receptor tyrosine kinase (RTK)-like molecules are involved in the proliferation and/or differentiation of alveolar type II alveolar epithelial response to injury. To test this hypothesis, we followed subtractive hybridization techniques and polymerase chain reaction procedures to isolate cDNAs encoding receptor tyrosine kinases from type II cells. This resulted in the isolation of a (i) novel cDNA fragment containing type III RTK-like motifs and ankyrin-like repeat motifs. Furthermore, (ii) we observed expression of this gene in epithelial cells, but nit in fibroblasts, and (iii) demonstrated its protein coding potential in in vitro translation systems. Preliminary studies also indicate that alterations in its expression in epithelial cells results in changes in cell morphology. These observations led us to believe that the product (p2.2) of this novel gene (EG-2.2) plays an important role in alveolar cell proliferation and differentiation. To test this hypothesis, we specifically propose to: I. Characterize the molecular nature of this newly identified gene (EG-2.2) and its protein product (p2.2). (1) The full length cDNA will be characterized and its localization on the human chromosome will be determined. (2) The cellular localization of the protein will be determined by immunocytochemical analyses. (3) The biochemical property of the encoded protein will be determined in in vitro autophosphorylation assays. II. Investigate the biological function of p2.2 (1) A chimeric protein containing the ligand binding domain of a known receptor and intracellular domains of p2.2, stably transfected in lung type II-like epithelial cells will be examined for its effects on cell proliferation and epithelial morphogenesis in collagen gels. (2) Association of proteins with the activated chimeric receptor will be assessed in immunoprecipitation experiments. (3) The expression of epithelial and also non-epithelial cell markers in these cells and in cells expressing sense or antisense 2.2-RNA will be investigated by Northern blot analysis and by immunocytochemistry. III. Investigate the expression of EG-2.2 in the rat lung. (1) Using PCR procedures we will isolate that rat homolog of human EG-2.2. (2) We will determine the expression of this gene and its protein product in normal rat lung tissue, and in the lungs of rats recovering after hyperoxic lung injury.
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