Conformational Regulation of Cell Surface Receptor Proteolysis

Abstract

Cell surface receptors can commonly undergo ectodomain shedding to modulate signaling pathways and cell contacts. To date, there are over 400 proteins that serve diverse functions on the cell surface that are predicted to undergo ectodomain shedding, and the shedding rates are commonly modulated in diseases such as cancer and inflammation. However, validating targets and understanding shedding regulation has been difficult due to a lack of control of proteolysis-inducing stimuli and unknown fates of cleavage products. Therefore, we built tools to help progress the field forward in understanding as well as investigated two cell surface receptors. The development of technology and tools to study proteolytically sensitive proteins is ongoing. Herein, two different tools that aid in the identification and study of mechanically sensitive proteins are described. We utilized HUH-tags to covalently link single-stranded DNA to target proteins of interest for single molecule force spectroscopy studies. We then developed an assay called SNAPS to identify switch-like proteins and screen for modulation of proteolysis by drugs. The SNAPS assay has innumerous applications in biotechnology and assessment of potential therapeutics, a few are described herein. We also investigated the biological impact of proteolysis on dystroglycan, an essential protein that forms a bridge between the extracellular matrix (ECM) and actin cytoskeleton. Proteolysis of dystroglycan is enhanced in disease states such as cancer, yet the biological impact of its proteolysis has not been elucidated. Moreover, there are muscular dystrophy disease-related mutations that map to the region where dystroglycan is cleaved by matrix metalloproteases (MMPs), but how they contribute to the mechanism of pathogenesis is not known. We have shown that disease mutations within the proteolysis domain of dystroglycan impact the overall conformation and stability of this domain, resulting in an increased in MMP cleavage. Proteolysis also has an impact on cell migration and cell morphology, suggesting that it may contribute to muscular dystrophy disease pathogenesis. Lastly, we turned our attention to Polycystin-1, a 7-pass transmembrane protein that is predicted to act as a mechanosensitive unit with Polycystin-2 in primary cilia. Its putative shedding is predicted to be functionally different than dystroglycan’s; instead of having a buried proteolysis site, its extracellular juxtamembrane region is proposed to involve dissociation of two non-covalently linked subunits. We investigated several disease-related mutations within its domain and performed initial tests for studying Polycystin-1 in the context of cultured cells.