Cell locomotion is a property, inherent to eukaryotic cells, that is critical to the development, homeostasis, repair and disease processes of multicellular organisms. The molecular mechanisms that regulate cell locomotion are poorly understood by hyaluronan (HA) and one of its cell binding sites, HARC (HA receptor complex) has been implicated in stimulating tumor cell locomotion. It is the long term objective of this proposal to investigate the molecular mechanisms by which HA:HARC interactions promote locomotion and to ultimately determine their role in physiological and disease processes. Our working hypothesis proposes that HA stimulates cell locomotion by activating such messenger mechanisms as protein tyrosine phosphorylation and that this effect is mediated by HARC which contains a hyaluronan binding protein (HABP) and a protein tyrosine kinase that is related to pp60c-src. HA:HARC interactions are predicted to act as an autocrine motility mechanism that regulates tumor cell locomotion. This hypothesis will be addressed by four specific aims: 1. What are the mechanisms by which HA promotes cell locomotion including binding of this polymer to cells, its effects on adhesion and its effect on protein tyrosine phosphorylation? 2. What is the functional role of HABP in mediating the above effects of HA? 3. What is the biochemical and molecular structure of the HABP associated with HARC? 4. What are the properties of the protein kinase associated with HARC? The first specific aim will be addressed by utilizing a fibroblast cell line that contains a promotable EJ-ras construct and that exhibits increased locomotion when the mutant ras oncogene is promoted. This increase is mediated by HA and HABP. Since sensitivity to HA:HABP can be modulated by modifying expression of the mutant ras gene, this model represents a unique opportunity to dissect the molecular mechanisms by which HA promotes tumor cell locomotion. To aid in assessing the role of HABP as a mediator of HA (Specific Aim #2), neutralizing monoclonal antibodies to HABP, that have been developed in the past year, will be used. To ultimately and directly test the working hypothesis and long term objective of defining the physiological role of HA:HARC interactions, it will be essential to characterize HABP and the associated protein kinase as indicated in specific aims 2 and 4. Characterization of HABP will be aided by a panel of monoclonal antibodies that have been prepared against native HABP and a partial cDNA encoding HABP. Because of its serological similarity to pp60c-src, the possibility that the protein tyrosine kinase associated with HARC is pp60c-src will be addressed by following the association of both metabolically-labelled c-src and genetically marked v- src (using temperature sensitive pp60v-src mutants) with HARC. Collectively these experiments will constitute the first critical step towards defining the role of HA:HARC in physiological and disease processes such as tumor progression. Definition of the mechanisms regulating cell locomotion will provide critical knowledge pertinent to both the genesis of tumor progression and other diseases that may lead to clinical application.
