Pamela Cowin - US grants

The long term goal of this project is to characterize the role of desmocollins in cell-cell adhesion. Desmocollins are major glycoprotein components of the desmosome, and the targets of adhesion-disrupting antibodies present in the sera of patients with the severe blistering disease pemphigus vulgaris. Structurally, they form a distinct subset of the cadherins, a family of cell-cell adhesion and recognition molecules. Desmocollins occur as a number of tissue-specific isoforms generated by alternative splicing. The precise roles of these isoforms are not known. It is hypothesized that they are essential to the adhesive mechanism of the desmosome and may play a role in cellular recognition, cytoskeletal binding and the reception and transduction of initial cell-cell contact signals. The distribution of the desmocollin isoforms in both adult tissues and during embryonic development will be examined immunochemically and by PCR techniques to determine whether the pattern of expression is compatible with a role in morphogenesis. The interactions of the isoforms with other junctional components and with kinases will be examined, by co-precipitation and in vitro binding assays, to identify whether they function in signal transduction and/or cytoskeletal binding. Similar techniques will be used to define whether their association in the plane of the membrane is homotypic or heterotypic. The sequences that mediate and regulate the binding affinity of these molecules will be studied by comparing the capacity of wild type and recombinant desmocollins to support cell-cell adhesion and the ability of adhesion disrupting antibodies to interfere with this process. The sequences that govern the specific targeting of desmocollins and cadherins to their neighboring but biochemically distinct membrane domains will be studied by examining the junctional localization of chimeras of these molecules. These studies should provide new insights into the specific role of the desmocollins in cell-cell adhesion as well as the pathogenesis of the blistering disease pemphigus vulgaris.

The long term goal of this project is to characterize the role of desmocollins in cell-cell adhesion. Desmocollins are major glycoprotein components of the desmosome, and the targets of adhesion-disrupting antibodies present in the sera of patients with the severe blistering disease pemphigus vulgaris. Structurally, they form a distinct subset of the cadherins, a family of cell-cell adhesion and recognition molecules. Desmocollins occur as a number of tissue-specific isoforms generated by alternative splicing. The precise roles of these isoforms are not known. It is hypothesized that they are essential to the adhesive mechanism of the desmosome and may play a role in cellular recognition, cytoskeletal binding and the reception and transduction of initial cell-cell contact signals. The distribution of the desmocollin isoforms in both adult tissues and during embryonic development will be examined immunochemically and by PCR techniques to determine whether the pattern of expression is compatible with a role in morphogenesis. The interactions of the isoforms with other junctional components and with kinases will be examined, by co-precipitation and in vitro binding assays, to identify whether they function in signal transduction and/or cytoskeletal binding. Similar techniques will be used to define whether their association in the plane of the membrane is homotypic or heterotypic. The sequences that mediate and regulate the binding affinity of these molecules will be studied by comparing the capacity of wild type and recombinant desmocollins to support cell-cell adhesion and the ability of adhesion disrupting antibodies to interfere with this process. The sequences that govern the specific targeting of desmocollins and cadherins to their neighboring but biochemically distinct membrane domains will be studied by examining the junctional localization of chimeras of these molecules. These studies should provide new insights into the specific role of the desmocollins in cell-cell adhesion as well as the pathogenesis of the blistering disease pemphigus vulgaris.

Desmogleins and desmocollins are the major adhesive components of desmosomes and form two distinct subtypes of the cadherin superfamily of cell-cell adhesion and morphoregulatory proteins. The adhesive properties and cytoskeletal connections of these and other cadherin-type proteins are modulated by their association with several cytoplasmic proteins. Plakoglobin physically associates with both desmogleins and desmocollins at the desmosome as well as with classical cadherins which concentrate in the adherens junction. As the only component to interact with all three of the major cell-cell adhesive proteins of epithelia, plakoglobin is uniquely positioned to play a key role in co-ordinating the adhesive properties of the cell. Plakoglobin is also the structural and functional homologue of a protein involved in cell fate determination in Drosophila and has been shown to participate in Wnt-l transformation of PC12 cells. Furthermore plakoglobin has recently been shown to form cytosolic complexes with another structurally related protein, APC, the product of a gene linked to an inherited form of colon cancer. This family of proteins may therefore be considered central players in cell-cell adhesion and tumor suppression. Given the involvement of plakoglobin in the important cellular processes outlined above I propose to further investigate the role of this protein and its receptors. My specific aims are: 1. To examine the effect on cell adhesion and proliferation of overexpressing native and mutant forms of plakoglobin. These mutants are designed to saturate specifically binding sites on cadherins , or, APC and to potentate or negate the interactions of these proteins with the cytoskeleton. 2. To identify the additional interactions of desmoglein/plakoglobin complexes: a) purify and clone a 70 kDa cytoplasmic partner of plakoglobin b) identify the novel partners binding to the cytoplasmic domain of desmoglein expressed as a fusion protein by screening libraries with the same fusion protein and employing the yeast two-hybrid system. 3) To characterize desmosomal cadherin receptors and functionally interfere with desmosome formation by use of an alkaline phosphatase tagged soluble ectodomain of desmoglein.

DESCRIPTION (Adapted from applicant's abstract): Our current hypothesis is that specific responses to Wnt signals are determined by both plakoglobin and beta-catenin in a manner dependent on their other protein associations and modifications. Preliminary results show that (1) transgenic expression of plakoglobin inhibits hair growth; (2) transgenic expression of beta-catenin produces tumors in mammary gland; (3) plakoglobin is glycosylated in the putative CSK-3beta phosphorylation site. Our aims address the following questions: (1b) does plakoglobin act antagonistically, synergistically or interchangably with beta-catenin to regulate cell growth in normal cells? K14-deltaN89beta-catenin mice will be created and their hair phenotype compared to the existing K14-deltaN80-plakoglobin mice. The spontaneous tumor incidence of existing MMTV-deltaN89beta-catenin and MMTV-deltaN80-plakoglobin mice will be compared. Antagonism or synergy will be tested in vivo by crossing of these pairs of mice and in vitro by examining cultured transgenic keratinocytes for the effects of expressing one transgene on the localization, stability and interaction of the produce of the other transgene. (1b) does up-regulated plakoglobin activate FGFR? K14-deltaN80-plakoglobin mice will be bred to FGF5-/- angora mice to look for counteraction of the long hair phenotype as evidence of intersection of the pathways. K14-deltaN80-plakoglobin trangenic keratinocytes will be examined for up-regulation of cadherins and for up-regulation of FGFs as potential FGFR ligands. (2) Does plakoglobin and beta-catenin up-regulation predispose epidermis to tumorigenesis? A panel of chemically induced skin tumors will be screened for up-regulation and relocation and mutations of plakoglobin and beta-catenin. K14 transgenic mice will be tested for enhanced or suppressed susceptibility to tumor induction by (a) breeding to mice expressing v-rasHa: (b0 transforming transgenic keratinocytes with v-rasHa and grafting to athymic mice; (c) standard chemical carcinogenesis protocols. (3) does glycosylation of plakoglobin play a role in protein stability, junction formation and Wnt-signaling? subcellular fractions of keratinocytes+/- junction formation and PC12 cells +/- wnt expression will be examined for presence and extent of plakoglobin glycosylaiton.

[unreadable] DESCRIPTION (provided by applicant): Funds are requested to support the Gordon Conference "Cell Contact and Adhesion" that will be held for the sixteenth time (30th year), June 8-13, 2002 at Proctor Academy, Andover, NH. The endurance of this conference testifies to its importance as a key meeting for the field of cell-cell interactions. It is interdisciplinary, encompassing molecular, structural, genetic and cellular approaches to dissecting the architecture of cell-cell contacts, determining their cellular, histological, and organismal roles and their involvement in human disease including cancer and developmental defects. The focus of this year's meeting will be the role of adhesive and occludens type cell-cell contacts in morphogenesis. The conference will interest molecular, cell, cancer and developmental biologists. The sessions cover areas of exciting and recent progress including: The role of cell contacts in: cell polarity, asymmetric cell division and planar polarity. Two sessions will cover the role of cell contacts in stem cell survival and cell fate determination. This theme has generated much excitement and will be explored in the vascular and cardiac system, epidermis and mammary gland. Three sessions will cover the role of cell contact proteins and their associated adaptor proteins in signal transduction processes governing proliferative control, cytoskeletal reorganization, cancer and metastasis. The roster of speakers includes established and renowned scientists who are active leaders in this field as well as young investigators and junior trainees who have recently contributed incisive data. The chairs have been chosen for their ability to generate discussion on a wide range of topics. As is traditional for this meeting, four slots have been reserved in the last session for the inclusion of late breaking developments and a short talk slot has been allocated at the end of each session to highlight the work of promising young investigators. [unreadable] [unreadable] This conference will bring together scientists working in vertebrate and invertebrate systems who do not meet in other forums. The components of cell-cell contacts are tumor suppressors and oncogenes and thus this conference will be highly relevant for the mission of the NCI. Cell-cell contact proteins provide the physical force that unite cells into functional tissues during embryogenesis and, as the genetic approaches highlighted in this meeting show, defects in these components lead to birth defects that are highly relevant to the mission of NICHD. This conference is also relevant to the mission of, and will report research that is also funded by NIDDK, NIAMS, NHLBI and NIGMS. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The purpose of this proposal is to study a novel uncharacterized GPCR. The hypothesis to be tested is that this orphan GPCR specifically demarcates activated epithelial stem cells and multipotent progenitors and critically regulates the unique proliferative characteristics of these cell populations. Preliminary analysis of the expression of a lacZ reporter knocked into the locus of the GPCR shows highly specific localization to well-characterized epithelial stem cell niches during embryonic and pubertal mammary and hair development. Aim 1 is designed to establish the physiological role of the GPCR. Characterization of the reporter expression pattern throughout the mammary cycle will be completed. Cells that express the reporter will be isolated by FACS and their stem/progenitor cell properties will be tested functionally. The regenerative capacity of reporter-positive cells will be tested in transplantation studies carried out at limiting dilution and their potency tested by analysis of the outgrowth phenotypes. Their relationship to other putative mammary stem/progenitor cell populations will be determined by FACS and immunohistochemical marker analysis. In aim 2 we will study the in vivo functional effects of loss of the GPCR signaling capability. The phenotypic effects of homozygous deletion of the GPCR cytoplasmic domain will be studied on mammary development and on murine mammary cancer models that arise from stem/progenitor cell expansion. The impact on stem/progenitor content, proliferative activity and tumor-promotion will be determined. These experiments are warranted by the interaction of GPCR with the tumor suppressor protein Discs large (Dlg), which regulates cell polarity, asymmetric division, proliferation and migration and by preliminary data indicating retardation in ductal outgrowth. PUBLIC HEALTH RELEVANCE: This novel orphan G-protein coupled receptor provides a highly specific marker of epithelial stem cells as they become activated to proliferate. As such it may have considerable clinical relevance as a prognostic marker of tumors from many different epithelial sources that arise from deregulated stem/progenitor cell proliferation. As a cell surface protein, reagents against this G-protein coupled receptor will provide powerful tools to identify, monitor and eliminate cancer-initiating cells that manifest stem cell-like behaviors such as the ability to undergo asymmetrical division. One quarter of all drugs in clinical trial target G-protein coupled receptors. Thus the development of antibodies and small molecule inhibitors against this protein could yield important translational initiatives to target breast cancer stem cells.