Our hypothesis is that keratinocyte specific G-alpha proteins play crucial roles in biologic processes that are critical to the normal responses and functions of these cells. Several biochemical pathways and systems that influence keratinocyte growth and differentiation have recently been elucidated and G proteins are signal transducing elements that interact with or mediate many of these pathways. The overall goal of this proposal is to identify and characterize both tissue specific and biologically important G-alpha genes from human keratinocytes with the following specific aims: 1) Identification of G-alpha genes from human keratinocytes. We have utilized a simple and rapid method of generating short G-alpha cDNA from keratinocytes via PCR by priming off highly conserved areas of G-alpha DNA sequences. An efficient strategy for cloning, screening by southern blotting and sequencing unique cDNAs has been established. Three G-alpha genes have been identified from 11 cDNA clones evaluated to date. Two of these represent members from the Gq class of G-alpha subunits and one is from the Gi class. Since keratinocytes play multiple roles in cutaneous biology, it is expected that several uniquely functioning critical G-alpha genes will be identified in this process. 2) Characterization of G-alpha genes via mRNA expression studies. We will test unique PCR generated G-alpha clones for tissue specificity and for restriction to proliferating or differentiating keratinocytes via northern analysis and in situ hybridization. Using these methods we have identified a keratinocyte specific Gq class G-alpha subunit, NHEK 13, not expressed in other cells cultured from the skin including fibroblasts, microvascular endothelial cells and melanocytes. 3) Molecular analysis of keratinocyte G-alpha gene expression. Selected G-alpha PCR generated cDNA segments will be used to screen a human keratinocyte cDNA library. cDNA clones will be sequenced and analyzed for conserved sequences and identity with other G- alpha genes, allowing biologic predictions for the cloned genes by comparing similarities with other G-alpha genes. Specific antibodies to keratinocyte restricted or novel G proteins will be obtained if available or produced for use in immunohistochemical staining and western blotting to evaluate the role that these G proteins play in keratinocyte biology. Using this strategy we have confirmed that NHEK 13 is homologous to G- alpha16 which was originally cloned from HL-60 cells and not expected to be present in keratinocytes. Our finding that a G-alpha homologue is expressed by human keratinocytes is highly significant and relevant to keratinocyte biology. These studies will provide an important scientific basis for the understanding of data delivery and processing within keratinocytes. Understanding these processes is directly relevant to developing new strategies for management of skin disease.
| Rock, B M; Xin, L; Wilcox, J N (1997) Two Gq class G proteins are expressed in human keratinocytes. J Invest Dermatol 109:645-9 |
