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The Star (steroidogenic acute regulatory protein)-related transfer (START) domain superfamily is characterized by a distinctive lipid-binding motif. START domains typically reside in multidomain proteins, suggesting their function as lipid sensors that trigger biological activities. Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is an example of a START domain minimal protein that consists only of the lipid-binding motif. PC-TP, which binds phosphatidylcholine exclusively, is expressed during embryonic development and in several tissues of the adult mouse, including liver. Although it catalyzes the intermembrane exchange of phosphatidylcholines in vitro, this activity does not appear to explain the various metabolic alterations observed in mice lacking PC-TP. Here we demonstrate that PC-TP function may be mediated via interacting proteins. Yeast two-hybrid screening using libraries prepared from mouse liver and embryo identified Them2 (thioesterase superfamily member 2) and the homeodomain transcription factor Pax3 (paired box gene 3), respectively, as PC-TP-interacting proteins. These were notable because the START domain superfamily contains multidomain proteins in which the START domain coexists with thioesterase domains in mammals and with homeodomain transcription factors in plants. Interactions were verified in pulldown assays, and colocalization with PC-TP was confirmed within tissues and intracellularly. The acyl-CoA thioesterase activity of purified recombinant Them2 was markedly enhanced by recombinant PC-TP. In tissue culture, PC-TP coactivated the transcriptional activity of Pax3. These findings suggest that PC-TP functions as a phosphatidylcholine-sensing molecule that engages in diverse regulatory activities that depend upon the cellular expression of distinct interacting proteins.

New Zealand obese (NZO/HlLt) male mice develop polygenic diabetes and altered phosphatidylcholine metabolism. The gene encoding phosphatidylcholine transfer protein (PC-TP) is sited within the support interval for Nidd3, a recessive NZO-derived locus on Chromosome 11 identified by prior segregation analysis between NZO/HlLt and NON/Lt. Sequence analysis revealed that the NZO-derived PC-TP contained a non-synonymous point mutation that resulted in an Arg120His substitution, which was shared by the related NZB/BlNJ and NZW/LacJ mouse strains. Consistent with the structure-based predictions, functional studies demonstrated that Arg120His PC-TP was inactive, suggesting that this mutation contributes to the deficiencies in phosphatidylcholine metabolism observed in NZO mice.

Phosphatidylcholines (PtdChos) comprise the most common phospholipid class in eukaryotic cells. In mammalian cells, these insoluble molecules are transferred between membranes by a highly specific phosphatidylcholine transfer protein (PC-TP) belonging to the steroidogenic acute regulatory protein related transfer (START) domain superfamily of hydrophobic ligand-binding proteins. The crystal structures of human PC-TP in complex with dilinoleoyl-PtdCho or palmitoyl-linoleoyl-PtdCho reveal that a single well-ordered PtdCho molecule occupies a centrally located tunnel. The positively charged choline headgroup of the lipid engages in cation-pi interactions within a cage formed by the faces of three aromatic residues. These binding determinants and those for the phosphoryl group may be exposed to the lipid headgroup at the membrane-water interface by a conformational change involving the amphipathic C-terminal helix and an Omega-loop. The structures presented here provide a basis for rationalizing the specificity of PC-TP for PtdCho and may identify common features used by START proteins to bind their hydrophobic ligands.