Abstract 412: Dissection of Thrombospondin-4 Domains Involved in Adaptive ER Stress Responsive Signaling and Secretory Pathway Functions

Abstract

Thrombospondins are a family of stress-inducible secreted glycoproteins with well-characterized roles in the extracellular matrix and tissue remodeling. We recently reported critical intracellular functions for thrombospondin-4 (Thbs4) by activation of an adaptive endoplasmic reticulum (ER) stress pathway in cardiomyocytes, in part by promoting activation of activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins and activation of this adaptive ER stress response, and determine the domains of Thbs4 involved in secretory pathway functions. We show that the N-terminal laminin G like domain (LamG) of Thbs4 exhibits intracellular localization almost exclusively at the Golgi apparatus and is robustly secreted in cultured cardiomyocytes, indicating rapid flux through the secretory pathway. We also generated a full length Thbs4 with mutations in calcium-binding motifs within the type III repeat (T3R) domain, including mutations homologous to human disease-causing mutations identified in THBS5/COMP. While wildtype Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, the Thbs4 calcium-binding mutant localized predominately to the ER and was not secreted. We identified BiP (Grp78) as a novel binding partner of Thbs4 in cardiomyocytes in vitro and in vivo, and utilized adenoviruses overexpressing Thbs4 domain mutants in cardiomyocytes to map the TSP-C domain (L-type lectin domain) of Thbs4 as the BiP-interacting motif. Additionally, the TSP-C domain of Thbs4 bound Atf6α and overexpression of the TSP-C domain alone was sufficient to induce the adaptive ER stress pathway in cardiomyocytes, activate Atf6α, and protect against ER stress-induced cell death. These studies characterize the functional domains of Thbs4 and aid in parsing out the intracellular and extracellular roles of thrombospondins. Taken together, data indicate critical functions for the TSP-C and LamG domains of Thbs4 in ER stress-responsive and secretory pathway functions, respectively.