Eukaryotic cells control the levels of their major membrane lipid, phosphatidylcholine (PtdCho), by balancing synthesis with degradation via deacylation to glycerophosphocholine (GroPCho). Here we present evidence that in both yeast and mammalian cells this deacylation is catalyzed by neuropathy target esterase (NTE), a protein originally identified by its reaction with organophosphates, which cause nerve axon degeneration. YML059c, a Saccharomyces cerevisiae protein with sequence homology to NTE, had similar catalytic properties to the mammalian enzyme in assays of microsome preparations and, like NTE, was localized to the endoplasmic reticulum. Yeast lacking YML059c were viable under all conditions examined but, unlike the wild-type strain, did not convert PtdCho to GroPCho. Despite the absence of the deacylation pathway, the net rate of Levels of phosphatidylcholine (PtdCho), 1 the major membrane lipid of eukaryotic cells, are tightly regulated by coordination of its synthesis and degradation. In both yeast (1) and mammalian cells (2), PtdCho synthesized by the CDP-choline pathway (see Fig. 1) is deacylated by as yet unidentified phospholipases to form glycerophosphocholine (GroPCho). In principle, this deacylation at both sn-2 and sn-1 positions of PtdCho could be mediated either by a single enzyme with phospholipase B activity or by sequential action of a phospholipase A2 and a lysophospholipase. We have reported that when mixed micelles of PtdCho with detergent were incubated with the recombinant catalytic domain of neuropathy target esterase (NTE), fatty acid was liberated very slowly from the sn-2 position followed by rapid deacylation of the resulting lysophospholipid (3). Because the rates and selectivities of bond cleavage observed in phospholipase assays in vitro are profoundly affected by the physicochemical nature of the substrate (4 -6), it is possible that in vivo NTE could deacylate the sn-2 position of PtdCho more efficiently than observed in our experiments. Thus, NTE might be the phospholipase B responsible for converting PtdCho to GroPCho (see Fig. 1).NTE was originally identified as the target site for those organophosphates that cause a paralyzing delayed neuropathy with degeneration of long nerve axons (7). In adult animals NTE is present in the nervous system and a variety of nonneural tissues (8). NTE is also widely expressed during fetal development (9). Studies on green fluorescent protein (GFP)-tagged NTE constructs expressed in COS cells indicate that NTE is anchored to the cytoplasmic face of the endoplasmic reticulum (10).Definitive evidence that NTE can convert PtdCho to GroPCho could be obtained by comparing PtdCho metabolism in wild-type and NTE-null cells. However, mice lacking NTE die by mid-gestation (11, 12), and fibroblasts from day 8 embryos can be cultured for only limited periods (12). On the other hand, a gene, YML059c, in the yeast, Saccharomyces cerevisiae, encodes a putative protein with substantial sequence homology to NTE. The availability of a YML059c-null mutant...

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.

Placental development and imprinting co-evolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes, IGF2 and IGF2R, code for the growth promoter insulin-like growth factor 2 and its binding inhibitor, mannose 6-phosphate/IGF2 receptor, respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes that lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that evolution of this ESE led to the fortuitous acquisition of M6P/IGF2R IGF2 binding that drew IGF2R into parental conflict prior to imprinting, that may have accelerated subsequent affinity maturation. † The sequence of molecular evolutionary events that established placental viviparity, genomic imprinting and parental conflict in mammals remain poorly understood (1) . Genomic imprinting occurs when expression of one allele of a diploid gene is silenced depending on the parent-of-origin, e.g. either from the father or the mother. Parental conflict over the distribution of resources to offspring has been supported by the observation of reciprocal imprinting of genes coding for the growth promoter Insulin-like growth factor 2 (IGF2), and the cation-independent mannose 6-phosphate/ IGF2 receptor (M6P/IGF2R or IGF2R) (2) . IGF2 and IGF2R are two of the approximately 80 genes imprinted in mammals, and two of the five genes (with INS, MEST/PEG1 and PEG10) imprinted in marsupials. So far, no evidence supports the existence of imprinting in monotremes despite the presence of a chorio-vitelline placenta (3, 4). On the basis of functional data, IGF2R transports M6P modified acid hydrolases to the pre-lysosomes (5). Of the 15 extra-cellular domains of IGF2R, domain 11 binds IGF2 in therians, and internalizes the ligand for degradation, whereas M6P bind to domains 3, 5 and 9 (5). Igf2 rescues placental dependent embryonic lethality associated with laboratory created murine parthenogenesis, implicating IGF2 supply as a regulator of placental development (6). Disruption of the maternal Igf2r allele results in Igf2 dependent overgrowth and fatality, supporting that IGF2R antagonizes the function of IGF2 (7,8). The structure of the unbound human domain 11 shows that the IGF2 binding site composed of defined loops (AB, CD, FG and HI, Fig. 1A and Fig. S1) but how this domain 11 evolved to bind IGF2, and the relationship to imprinting co-evolution, remain unknown (9-12).We established a high resolution structure of the human IGF2R:IGF2 complex and then compared this to other phylogenetically informative vertebrates. We adopted an NMR approach as the side chain amino acid interactions across the binding interface were not resolved in our 4.1Å resolution co-crystal structures (9). Wild-type human domain 11 and IGF2 failed to form a stable association in initial NMR studies. However, we ...