Our long-term objectives for this project are to obtain an understanding of the control mechanisms involved in regulating human lymphohematopoietic stem (LHSC) and progenitor cells. This is an important area of research because of the potential clinical impact of being able to manipulate this process. We have taken the approach that receptor tyrosine kinases are likely to be important in regulating the cell numbers or the development of these cells. This premise is based on the accumulated evidence of their role in both hematopoietic and nonhematopoietic cells. We have successfully cloned the cDNA for a human receptor tyrosine kinase, STK-1, that is most closely related to c-fms and c-kit, both growth factor receptors that are involved in hematopoiesis. STK-1 was cloned from a population of human bone marrow cells that are greatly enriched for LHSC and progenitor cells. Initial studies indicate that the expression of STK-1 is restricted to this """"""""young"""""""" fraction of bone marrow. The mouse homolog of this receptor was cloned recently and was shown to have similarly restricted pattern of expression. The receptor is therefore of interest because of the potential insights it may give us into the control of hematopoiesis. We have proposed a series of experiments for which most of the reagents are already available as an outcome of our preliminary work. We will carefully examine the expression of STK-1 in subfractionated human marrow by RT-PCR to determine in which cells STK-1 is likely to play a role. The fractions tested will include some of the most highly purified LHSC, to give us an indication of whether the receptor is expressed on these cells. Antibodies to the receptor will allow us to confirm these results by FACS analysis and may enable the further purification of these cells. We will investigate the regulation of the gene coding for STK-1. We will first complete the cloning of the region containing the gene. We will search for cis-acting sequences that confer expression by use of a reporter gene assay. Thus insight may be gained into the control of other genes with expression similarly restricted to """"""""young"""""""" hematopoietic cells. We will analyze the pathway by which STK-1 signal transduction takes place. We will look at the targets of signal transduction already identified for other receptors and hope to find some novel proteins as well. Lastly, we will test the hypothesis that this receptor is involved in malignancy or other disease processes involving the hematopoietic system. The finding that STK-1 is expressed in several leukemic cell lines may be an indication that it is somehow involved. RNA and DNA samples from patients afflicted with these diseases will be tested over-expression or altered expression of the receptor.
