Project Summary: Praline is a multifunctional imino acid with myriad uses in the cell. Aside from direct incorporation into protein, praline can be metabolized via a process known as the praline cycle. Here, praline is oxidized by praline oxidase (PRODH) to form L'.11-pyrroline-5-carboxylate (P5C). PRODH is a FAD+ dependent enzyme that donates electrons to complex II of the mitorchondrial electron transport chain thus coupling praline oxidation to ATP synthesis. P5C is converted back into praline by the NADH dependent enzyme pyrroline-5-carboxylate reductase (PYCR) to provide reducing power for glycolysis and the pentose phosphate pathways. It is unknown how osteoblasts obtain praline, how praline uptake is regulated, or if and when the praline cycle is required during differentiation. Osteoblasts express a diverse array of membrane-tethered amino acid transporters to facilitate praline uptake. We have identified the system A neutral amino acid transporter SNAT2 (encoded by Slc38a2) as the most highly expressed putative praline transporter in osteoblasts. Our preliminary data indicates WNT stimulates praline uptake through SNAT2 that is necessary for osteoblast differentiation in vitro. Moreover, mice homozygous for a null allele of Slc38a2 (Slc38a2"1 -) have defects in endochondral ossification. In this proposal, we will 1) establish the necessity of praline uptake through S/c38a2/SNAT2 to regulate osteoblast differentiation and bone formation in vivo, 2) determine how SNAT2 activity is regulated by WNT signaling and 3) elucidate the necessity of praline metabolism via the praline cycle in differentiating osteoblasts. Our findings will have broad implications in bone development, maintenance of bone mass, skeletal repair and regeneration.