Steven A. Rosenzweig - US grants

The long-range goal of this proposal is to characterize the structural and functional properties of the cholecystokinin (CCK) receptor in pancreatic acinar cells using biochemical and immunochemical techniques. In biochemical studies, CCK binding proteins will be labeled in intact acinar cells and isolated membranes with a new photoaffinity analog of CCK-8, as well as by chemical cross-linking protocols. The potential multicomponent nature of these labeled proteins will be analyzed by gel filtration chromatography and sucrose density gradient centrifugation. Using the recently obtained information that the affinity labeled CCK binding proteins are glycoproteins, the combined techniques of lectin affinity chromatography, CCK affinity chromatography and HPLC gel filtration will be used to isolate CCK receptors from detergent extracts of rat pancreatic membranes for detailed structural analysis. Nearest neighbor analysis of the CCK binding proteins to one another and to other membrane proteins will be performed as will a structural comparison of the CCK receptor in brain to that in pancreas. The role receptor oligosaccharide side chains may have in CCK binding or action will also be examined. For immunochemical studies on the CCK receptor, polyclonal and monoclonal antibodies will be raised against purified CCK receptors. Structural studies will include analysis of the potential multicomponent nature of the receptor, studies on the biosynthesis of receptor and the application of these antibodies toward purification of receptor by immunoaffinity chromatography. Studies on receptor function will be directed at examining the effects of anti-receptor antibodies on CCK binding and/or action. Whether the anti-receptor antibodies directly mimic or antagonize CCK action on intact pancreatic acini will be examined as will the covalent modification of the CCK receptor via phosphorylation. These studies should greatly enhance our understanding of the CCK receptor at the molecular level and provide insight into the role CCK plays in the regulation of pancreatic function and satiety.

The long-range objective of this proposal is to characterize the structural and functional properties of the cholecystokinin receptor on pancreatic acinar cells and retinal neuronal cells. Comparative studies will be directed at examining the affinities and molecular weights of the CCK receptors on rat pancreas and toad retina by competition binding analyses and affinity labeling techniques. Functional comparisons of these receptors will be obtained by monitoring the relative effectiveness of CCK-8 to stimulate 45Ca2+ efflux and polyphosphoinositide turnover in intact pancreatic lobules and retinal monolayers, respectively. We will also compare the pancreatic and retinal CCK receptors using specific antibody probes. To this end, a major emphasis has been placed on obtaining specific polyclonal antibodies to the Mr 81 K CCK recognition subunit of the receptor isolated from rat pancreatic plasma membranes. Rat pancreatic plasma membranes will be solubilized with Nonidet P-40 and the Mr 81 K protein will be purified by a number of sequential chromatographic steps followed by SDS gel electrophoresis. It will then be electroeluted from gel slices and either immobilized onto nitrocellulose paper and used to immunize rabbits or electroblotted onto activated glass for NH2-terminal microsequence analysis on a gas-phase sequenator. In the latter case, we will obtain the sequence of the first 10-20 NH2- terminal amino acid residues of this protein and with this data, have mg quantities of the corresponding peptide synthesized. Synthetic peptide will then be used to immunize rabbits and obtain site-specific antibodies to the CCK receptor. Using the specific antibody probes generated, we will complement our comparisons of the pancreatic and retinal CCK receptors by Western blotting analysis and immunoprecipitation of biosynthetically labeled receptor. We will also determine whether the CCK receptor in pancreas undergoes phosphorylation on tyrosyl residues in response to CCK binding or whether it undergoes regulatory phosphorylation by agents such as protein kinase C. These studies will provide considerable information, at the molecular level, of CCK receptor structure and function and will greatly enhance our understanding of the role CCK play in the regulation of pancreatic and retinal function.

The long range objective of this proposal is to analyze the structure and function of the receptors for vasoactive intestinal peptide (VIP) and insulin in retinal and hepatic membranes. Comparative studies will be directed at examining the binding specificities and kinetics of binding of the VIP receptors in membranes prepared from bovine retina and liver and rat retina and liver. Structural comparisons of the VIP receptor in these tissues will be accomplished with the use of affinity labeling methodologies. Affinity labeled receptors will be analyzed by SDS gel electrophoresis and autoradiography of the dried gels. Similarly, analysis of the kinetics of binding of the insulin receptor present in neural retina and retinal endothelial cells will be carried out and compared to its peripheral counterpart in liver. VIP receptor function in retina and liver homogenates and cells will be determined by measuring VIP's ability to activate adenylate cyclase, cause alterations in Ca2+ fluxes and modulate polyphosphoinositide turnover in these two tissues. This analysis will provide information regarding whether the VIP receptor can interact with more than one signal transduction system in the retina. Isolation and purification of the retinal VIP receptor will also be undertaken in order to gain detailed structural information about this protein. Structural analysis of neural retina insulin receptor alpha and beta-subunits will be carried out both on affinity labeled and purified subunits. Affinity labeled VIP and insulin receptors will be treated with endo-beta-glucosaminidase F to remove N-linked oligosaccharide chains in an effort to determine if the polypeptide backbones of these proteins are the same across tissues and thus modified post-translationally via glycosylation or represent different gene products. Similarly, these receptors will be treated with endo-beta-glucosaminidase H, O-glycanase and neurminidase in order to obtain information on differential glycosylation of these receptors in different target tissues and thus the potential role differential glycosylation plays in altering receptor function. In the case of the insulin receptor, functional studies will include analysis of beta-subunit autophosphorylation in response to insulin. The effect of experimental diabetes on insulin receptor expression in rat retina will also be determined. The analyses planned will be instrumental in establishing functional roles for VIP and insulin receptors in the neural retina as well as providing insight into their structural characteristics.

The insulin-like growth factor 1 (IGF-1 receptor (IGF-1R) is an essential regulator of cell growth and transformation. IGF-1 and IGF-2, via the IGF-1R, are potent breast cancer cell mitogens that promote the tumorigenic potential of cancer cells. The objective of this proposal is to develop reagents that block IGF-1R signaling. The insulin-like growth factor binding proteins (IGFBPs) bind IGF-1 and IGF-2 with higher affinities than the IGF-1R and serve to both protect the IGFs from degradation and reduce their delivery to the IGF-1R. The hypothesis of the proposal is that blockade of IGF-1R activity can be accomplished by developing IGF antagonists based on the structure of the IGF binding domain on the IGFBPs. The goal of Aim 1 is to define the structure of the IGF-binding domain on rhIGFBP-2 using photoaffinity labeling and mass spectrometric analyses. To this end, IGF-1 derivatized with photactivatable groups within its IGFBP-binding domain will be synthesized. This will allow the precise identification of the sites of interaction between rhIGFBP-2 and IGF-1. Based on these analyses, deletion, truncation and site-directed mutants of IGFBP-2 will be generated and tested for IGF binding activity. Aim 2 will characterize the structure and function of a 15.8 kDa fragment of IGFBP-2. The goal of Aim 3 is to examine the mechanism responsible for IGFBPP-2 inhibition of IGF action. The goal of Aim 4 is to employ phage display to screen libraries for peptides having a high affinity for the IGFBP-binding domain on IGF-1 as an alternative means of designing IGF antagonists.

DESCRIPTION (provided by applicant): The insulin-like growth factor 1 receptor (IGF1R) is an essential regulator of cell growth and transformation that promotes tumor cell proliferation, motility and protection from apoptosis. We have shown that in head and neck squamous cell carcinoma (HNSCC) cells IGF1R activation increases vascular endothelial growth factor (VEGF) synthesis and secretion, initiating an autocrine VEGF:VEGFR2 signaling loop. Our overall hypothesis stipulates that VEGF signaling via VEGFR2, leads to enhanced HNSCC tumorigenicity and invasive cell behavior. In strong support of this hypothesis we have identified a cluster of proteins involved in cell motility activated by VEGF stimulation. These proteins include human enhancer of filamentation1 (HEF1), cortactin, paxillin, and focal adhesion kinase. Of significance, HEF1 (or neural precursor cell expressed developmentally down-regulated 9/NEDD9) has been identified as a signature protein required for metastasis in melanoma and glioblastoma. Consistent with this, we have shown that VEGF stimulated migration, invasion and invadopodia formation are all dependent upon HEF1 expression. We will further define the role HEF1 plays in HNSCC invasive behavior in the following aims. The goal of AIM 1 is to define the mechanism through which HEF1 mediates VEGF induced HNSCC cell migration and invasion. Specifically, we will define the specific tyrosine residues in HEF1 that are phosphorylated in response to VEGFR2 activation using complementary biochemical techniques and site-directed mutagenesis. We will further define the effector(s) that bind to these sites of regulation and define the role of the N-terminal SH3 domain and its interacting proteins in invasion. In AIM 2 we will define VEGF regulation of invadopodia formation and the structural and functional contributions of HEF1 to this process. As a subset of this aim we will examine HEF1 localization to invadopodia, the role of its SH3 domain in this process and in matrix metalloproteinase delivery to invadopodia. In AIM 3 we will test the hypothesis that elevated HEF1 expression is prognostic for advanced stage HNSCC tumors in contributing to metastasis and underlying a poor prognosis. Five year survival studies will also be conducted. Human HNSCC tissue arrays and tumor/normal pairs will be examined for HEF1 expression and select activation of signaling pathways. We will also test the contribution of HEF1 to metastasis using mouse carcinogenesis and xenograft models. These studies will provide important evidence demonstrating a role for HEF1 in HNSCC metastatic signaling downstream of crosstalk to VEGFR2. This will lead to the development of novel strategies and therapeutic agents aimed at reducing the tumorigenic and metastatic effects of the IGF and VEGF pathways in HNSCC.