Galectins are a family of lectins which share similar carbohydrate recognition domains (CRDs) and affinity for small ␤-galactosides, but which show significant differences in binding specificity for more complex glycoconjugates. We report here the x-ray crystal structure of the human galectin-3 CRD, in complex with lactose and N-acetyllactosamine, at 2.1-Å resolution. This structure represents the first example of a CRD determined from a galectin which does not show the canonical 2-fold symmetric dimer organization. Comparison with the published structures of galectins-1 and -2 provides an explanation for the differences in carbohydrate-binding specificity shown by galectin-3, and for the fact that it fails to form dimers by analogous CRD-CRD interactions.Galectin-3 is a member of the galectin family of lectins defined by a conserved ϳ14-kDa carbohydrate recognition domain (CRD) 1 showing affinity for ␤-galactosides (1, 2). Abundantly expressed in a few cell types, such as macrophages and polarized epithelial cells in adults (2, 3) and others during embryogenesis (4), it tends to be localized in the cytoplasm and the nucleus. Although functions for galectin-3 have been proposed in each of these subcellular locations (5-7), it is also secreted by a nonclassical pathway (8, 9) and is found on the cell surface and in the extracellular matrix. There it binds and cross-links selected carbohydrate-containing ligands (10,11) and is thought to modulate cell adhesion (12-14) and cell signaling (15, 16). Many groups are currently studying the roles and uses of galectin-3 in cancer, inflammation, hostpathogen interaction, and nerve injury, among others (17, 18).Galectin-3 is unique among the known galectins in that, in addition to the canonical CRD (located at the C terminus), it contains an unrelated, non-carbohydrate-binding N-terminal domain of between 120 (in human) and 166 (in dog) amino acids (3,19). In contrast, galectins-1 and -2 are homodimers composed of the CRD alone, while galectins-4, -6, -8, and -9 possess an N-and C-terminal CRD linked in tandem by a short polypeptide segment (18). The galectin-3 CRD shows sequence identity ranging from 30 -40% with galectins-4 through -10, to 20 -25% with galectins-1 and -2. It has an affinity for lactose (Lac, K d ϭ 1 mM), and N-acetyllactosamine (LacNAc, K d ϭ 0.2 mM) similar to that of other galectins, but has a distinct profile for larger oligosaccharides (20, 21), including polyNAc-lactosaminoglycan, a polymer of ␤(1,3)-linked LacNAc units found on many extracellular matrix and cell surface molecules.Intact galectin-3, but not its CRD alone, shows avidity for multivalent glycoconjugates (10, 11), modulates cell adhesion (14), and induces intracellular signals (15). Thus it is thought that the N-terminal domain of galectin-3 promotes the formation of dimers or higher order oligomers, thereby permitting multivalent interactions essential for its biological activities.We report here the x-ray crystal structure of the CRD of human galectin-3 in complex with Lac and LacNA...

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Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for ∼50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1 degradation becomes essential when cells differentiate into meiosis. These results suggest that Ddb1, along with Cullin 4 and the signalosome, constitute a major pathway controlling genome stability, repair, and differentiation via RNR regulation.[Keywords: Genome stability; meiosis; ribonucleotide reductase; Ddb1; S. pombe] Supplemental material is available at http://www.genesdev.org.

Mammalian phospholipase D (PLD) activity becomes upregulated when cells are stimulated by a variety of hormones, growth factors, and other extracellular signals. Two distinct PLDs, PLD1 and PLD2, have been identified. The mechanism through which each PLD is activated, however, is poorly understood. Using transiently transfected human embryonic kidney fibroblasts (HEK293), we demonstrate here that PLD1 activity, and to a lesser extent PLD2 activity, is stimulated in response to epidermal growth factor (EGF). PLD2, but not PLD1, associates with the EGF receptor in a ligand-independent manner and becomes tyrosinephosphorylated upon EGF receptor activation. Tyrosine 11 (Tyr-11) of PLD2 was identified as the specific phosphorylation site. Mutation of this residue to phenylalanine enhanced basal activity almost 2-fold, but did not alter the magnitude of the EGF-mediated increase in PLD2 activity. In conclusion, we show here for the first time agonist-stimulated activation of both PLD1 and PLD2 in vivo and provide evidence of a distinct type of interaction for each isoform with the EGF receptor. Moreover, our results suggest that agonist-induced tyrosine phosphorylation plays a role in PLD2 regulation.