Investigation of the creatine biosynthesis negative feedback loop via creatine mediated control of arginine:glycine amidinotransferase expression in human HAP1 cells implies an intracellular creatine sensing mechanism

Abstract Cellular homeostasis of creatine (CT), integral part of the energy buffering and -transducing system connecting intracellular sites of ATP production and -utilization, comprises of mechanisms that increase CT, i.e., biosynthesis and cellular uptake, and CT-lowering processes, such as export and non-enzymatic conversion to creatinine. The negative feedback loop by which CT controls its biosynthesis via suppression of the rate-limiting enzyme arginine:glycine amidinotransferase (AGAT) is not well understood. We have used CRISPR to tag the C-terminus of AGAT with a nanoluc luciferase (NLuc) reporter in HAP1 cells. A biphasic decay of AGAT-NLuc in response to increasing extracellular CT was observed, whereas the decrease in AGAT-NLuc expression was directly proportional to the rise in intracellular CT levels with an approximate IC50 of 1–2 mM. CRISPR generated HAP1 CT transporter (CrT) null cells and HAP1 CrT null cells stably expressing a CrT-GFP fusion protein further demonstrated that the biphasic response to extracellular CT is mediated by a high-affinity (Km 9–10 µM) CrT dependent, saturable mechanism and a CrT independent, unsaturable uptake process. The direct response to intracellular CT suggests the existence of an intracellular CT sensing system enabling a dynamic cell response to changing CT concentration that is relevant for cellular CT homeostasis.