Proteolytically activated receptors (PARs) are members of a larger family of seven transmembrane segment G protein- coupled receptors, activated by proteolytic cleavage at specific bonds within their N-terminal extensions. PARs have previously been shown to be mediators of cell proliferation, and murine studies suggest a fundamental role for PARs in development. Work from this and other laboratories have identified PAR-3, which is preferentially expressed in hematopoietic tissue and magakaryocytes- platelets. Disparate results between platelet and megakaryocyte expression suggest that PAR-3 may be developmentally regulated during magakaryo- cytopioiesis. Genetic linkage analysis using radiation hybrid mapping panels localized the PAR-3 gene to the PAR gene cluster at 5q13, a site at or contiguous to the common proximal breakpoint in 5q- syndrome, an entity characterized by dysmegakaryocytopoiesis. Preliminary data using long-range PCR and Southern blot analysis of DNA from 5q-patients suggest a deletion or rearrangement involving PAR-3, making it a candidate gene for the disordered megakaryocytic differentiation seen in these patients. This proposal will establish a causative link between the PAR-3 gene and the 5q- syndrome. To characterize the genetic abnormality in 5q- syndrome, a cosmid contig will be constructed as a sequential representation of the critical interval along chromosome 5. Southern blot and FISH analysis will be completed to specifically evaluate molecular genetic defects within this region of the genome. Confirmatory in situ hybridization and immunohistochemical studies will be adapted to study PAR-3 expression patterns in subpopulations of hematopoietic cells. Should a definitive role for PAR-3 not be confirmed, the identification of other candidate genes within the deleted interval will be pursued. Confirmation of the candidate gene will be via complementation of normal and 5q- stem cells using adeno - adeno-associated (Ad-AAV) viral hybrid vectors. A HEL cell model will be developed for studying the molecular mechanisms of PAR-3 functions in megakaryocytopoiesis. If PAR-3 is causally related to 5a- syndrome, it will be the first demonstration of genetic defect in a PAR, and evidence that proteases may regulate hematopoietic cell differentiation and development. During the award period, the candidate will develop knowledge of genetics, gene therapy and basic science research in a laboratory that is well-equipped for the execution of this proposal. The candidate's long-term goal is the pursuit of a career in academic medicine.
| Gnatenko, Dmitri V; Cupit, Lisa D; Huang, Emily C et al. (2005) Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype. Thromb Haemost 94:412-21 |
