Vasculogenesis and angiogenic remodeling are basic requirements for organogenesis and wound healing. For vasculogenesis to occur, the endothelial cell progenitors must assemble properly. The current proposal will further define the molecular processes that direct renal microvascular endothelial targeting and assembly. The proposal hypothesizes that renal and other microvascular endothelial cells discriminate appropriate partners for vascular structure assembly based on signals they receive upon juxtacrine contact during directed migration. In this regard, previous studies by this investigator have shown that the ELK subclass of Eph family of receptors are critical in cultured human renal microvascular endothelial cells (HRMEC) attachment and assembly behavior. Further, the investigator has shown that the signals of the ELK receptor depend upon juxtacrine presentation of their ligands in variably oligomerized states on the surfaces of the contacting cells.
Three aims will be addressed. The experimental approach will involve traditional biochemical affinity studies as well as yeast-two hybrid system and use of dominant negative protein interactions. The first specific aim, to define the molecular basis for LERK-2 oligomerization, will be accomplished using two complementary approaches. The first will employ a yeast two-hybrid screen utilizing the cytoplasmic domain of LERK-2 as bait to screen a murine embryonic library. This study anticipates finding proteins that interact with the cytoplasmic domain of LERK-2. The complementary approach will involve standard cell culture labeling experiments to provide information about the cellular distribution about LERK-2 in stimulated and unstimulated cells. Subsequently, other experiments will utilize a LERK-2 cytoplasmic domain created as a GST fusion protein to allow binding to GSH-beads. This fusion protein will then be used to capture potential interacting proteins from the cytosol of stimulated cells. Finally, a series of chemical cross-linking studies and gel filtration electrophoresis experiments will be employed to investigate the state of oligomerization of LERK-2 following stimulation of endothelial cells. The second specific aim will focus on ELK signaling and, specifically, will investigate the molecular basis for the interaction between ELK and LMP-PTP, which was demonstrated by previous studies in this investigator's laboratory. In this regard, this laboratory has accumulated a wide range of experimental tools to address this interaction and will use the yeast two-hybrid system and baculovirus expression system to address these issues in addition to transient transfection assays and a dominant negative approach to examine these interactions. The third specific aim is a descriptive study that will determine the type of integrin present on cell lines used in this laboratory, using standard FACS analysis. In addition, transfected cell lines will be established to examine the interaction among integrins and signaling by ELK-LERK-2 interactions. This is a proposal which seeks a further 5 years of funding for the study of capillary morphogenesis by renal microvascular endothelial cells. The project is now in its fourth year, and has provided novel insights into cell-cell recognition events. In brief, it was learned that the ELK-LERK-2 ligand-receptor pair is present in renal endothelial cells during development, and that LERK-2, when presented as tetramers to ELK can stimulate precapillary cord formation in vitro. In contrast, dimeric LERK-2 only stimulates cell adhesion and migration in a matrix-specific fashion. Proteins that associate with the cytoplasmic tail of activated ELK have been identified, and while Grb-2 and Grb-10 associate with ELK stimulated by dimeric LERK-2, a tyrosine phosphatase LMW-PTP only associates with the receptor when it binds clustered, tetrameric LERK-2. It is therefore proposed that ELK and LERK-2 are counter-receptors on renal microvascular endothelial cells which cluster during cell-cell contact, and that both, ELK and LERK-2 serve to signal intracellular events which stimulate capillary cord formation. The approaches proposed for next term of the project are to use the yeast two hybrid system extensively, and to study in vitro interactions with GST fusions of ELK and LERK-2 (and mutants thereof) with the aim of identifying proteins that interact with the cytoplasmic tail of LERK-2 (aim 1) and ELK (aim 2). For ELK, this approach has been partially successful and aim 2 therefore seeks to further define signaling events that stimulate cell assembly by studying specific mutants of ELK and its partners, their mutual interactions, and their potential effects on cell assembly. Since dimeric LERK-2 stimulates cell adhesion and migration as well as ELK tyrosine phosphorylation, and since Grb-2, Grb-4, and Grb-10 have been found to associate with ELK upon stimulation with dimeric and tetrameric LERK-2, aim three is designed to assess the influence of LERK-2-ELK on integrin function, the involvement of small G-proteins in stimulating adhesion, cytoskeletal reorganization and migration, and activation of the JNK/SAP kinase pathway.
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