The purpose of this proposal is to continue studying the regulation and function of tissue (renal) kallikrein. Tissue kallikreins, a subgroup of closely related serine proteinases, have diverse functions depending on their location and substrate specificity. The best recognized biological role of tissue kallikreins is to produce vasoactive kinin peptides from kininogen substrates by limited proteolysis. Several members of this multigene family are involved in processing polypeptide hormones and growth factors. Advances in this and many other laboratories during recent years have made it possible to initiate a major effort to investigate the function of tissue kallikrein in hypertension and peptide- hormone processing. We have established technologies and developed an array of biochemical, immunological and molecular reagents which have paved the way for the currently proposed studies. Our goals will be accomplished by: (1). Purification and structural analysis of kallikrein gene family members from rat and human. Substrate specificities of kallikreins will be correlated with their 3-dimensional structures determined by X-ray crystallography and specific cleavages of polypeptide precursors by kallikreins will be defined. (2). Analysis of the structure and organization of rat and human kallikrein gene families. Sequence information will be used to design specific oligonucleotides for the identification of new kallikrein genes and their transcripts. (3). In vitro analysis of renal kallikrein gene regulatory elements. A rat renal kallikrein gene 5' promoter region spliced to a reporter gene will be used to define both cis- and trans-acting elements which regulate renal kallikrein gene transcription. (4). Analysis of renal kallikrein gene expression in vivo and identification of sequences essential for renal expression by transgenic techniques. The impact of elevated levels of circulating tissue kallikreins on the cardiovascular system will be assessed through the induction of kallikrein gene expression in transgenic mice. (5). Analysis of the genetic basis of kallikrein function in blood pressure regulation by defining the molecular defect in the kallikrein gene linked to RFLPs and the hypertensive phenotype in spontaneously hypertensive rats (SHR). (6). Inactivation of the mouse renal kallikrein gene by homologous recombination in embryonic stem cells. The potential roles of kallikrein in regulating blood pressure and processing bioactive peptides will be examined in the kallikrein-deficient mice. These studies will lend insight into the roles this group of fascinating enzymes play in human cardiovascular physiology and diseases.
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