SUMMARY Melanosome pH controls pigmentation and affects skin cancer risk; however, the signaling pathways that affect this important pigment mechanism are poorly understood. The Melanocortin 1 Receptor (MC1R), through transmembrane adenylyl cyclase (tmAC)-defined cAMP signaling pathways, has an important role in pigmentation, and affects skin cancer risk by activating the expression of key pigment synthesizing enzymes. But whether MC1R signaling affects melanosome pH has remained unclear. We recently identified a new cAMP signaling pathway in melanocytes, defined by the soluble adenylyl cyclase (sAC), that regulates melanosome pH. Whereas elevation of tmAC-dependent cAMP increases eumelanin by upregulating key pigment enzymes (e.g., tyrosinase), a reduction in sAC-dependent cAMP also increases eumelanin by inducing the alkalization of melanosome pH and enhancing tyrosinase activity. Thus, our overarching hypothesis is that sAC and tmACs regulate distinct cAMP signaling cascades in melanocytes and function in concert to control pigmentation. What remains unclear are the upstream and downstream mechanisms that control sAC-dependent regulation of melanosome pH and pigmentation. In our first Aim, we will use human primary melanocytes expressing either wild type MC1R or MC1R polymorphisms along with Mc1re mouse melanocytes and a novel Mc1re (e/e) conditional sAC knockout mouse to determine the interplay between MC1R signaling and sAC-dependent control of melanosome pH and pigmentation. In our first Aim, we will also assess whether bicarbonate, a known stimulator of sAC that has been linked to melanin synthesis, affects melanosome pH and pigmentation in human and mouse melanocytes in a sAC-dependent manner. In Aim 2, we will determine how sAC regulates melanosome pH and pigmentation. Our preliminary data suggests that sAC activates the cAMP effector protein exchange protein activated by cAMP (EPAC), which then stimulates the melanosome ion channel two-pore channel 2 (TPC2). Using genetic and pharmacological methods in mouse and human melanocytes, we will establish which EPAC isoforms and melanosome channels are required for sAC-dependent control of melanosome pH. Finally, our preliminary data suggest that sAC inhibition rescues the defective melanosome pH and tyrosinase activity in Oca2 deleted mouse melanocytes both in vitro and in mice. Thus, pharmacological sAC inhibitors are potential therapeutics for oculocutaneous albinism type 2. We will further explore this therapeutic possibility with a new conditional sAC knockout Oca2-/- (p/p) mouse model. Overall, the experiments in this proposal will systemically examine the cAMP dependent signaling cascades that regulate melanosome pH and pigmentation. The proposed studies will establish new models that will overcome limitations in our investigation of cAMP signaling in pigmentation, will provide greater insight into the cAMP-dependent mechanisms that control melanosome pH, and may lead to new therapeutic...