Elucidating the molecular regulation of superdark transmembrane protein 184C (TM184C)

Using structure-to-sequence predictions, our lab recently identified TM184C as a previously uncharacterized ‘superdark' GPCR-like protein. We have shown that this protein is highly enriched in cellular projections and facilitates cargo transfer through microtubule-based intercellular connections. Alongside, we have also shown that TM184C controls the formation and activity of autophagosomes leading to constrained autophagy. Furthermore, our results provide preliminary insights into why TM184C is a poor prognosticator in cancers such as glioblastoma, pancreatic cancer, and ovarian cancer. As such, we are now trying to understand the different modes of TM184C regulation and how they relate to cancer biology.We have shown that TM184C functions primarily in vesicles with acidified lumen, including autophagosomes, late endosomes, and lysosomes. This led us to hypothesize its spatiotemporal function may be partly regulated by pH. To address this question, we performed a structural analysis to identify residues that may confer pH regulation of TM184C. We stably expressed these mutant residues in HEK293A cells as the non-cancerous model, and Panc-1 cells, as the pancreatic cancer model. With the use of live cell confocal fluorescence microscopy, we observed that these mutants produce striking cellular phenotypes, including abnormal nucleus and vesicles, consistent with hyperactivation or loss of function, supporting a model in which TM184C responds directly to vesicular acidity. Our mutational studies thus strongly indicate that TM184C is indeed regulated by vesicular pH, suggesting that any diseases or drugs that alter vesicular pH may modulate TM184C function. Our research has important implications in cancer biology since disrupted pH and autophagy are two of the key components that tumor cells exploit to resist therapy and adapt to the tumor microenvironment.