Purification, identification, and characterization of an osmotic response element binding protein.

Kidney cells, especially the epithelial cells lining the collecting tubules in the inner medulla, are constantly exposed to concentrated urine. They are protected from the osmotic effect of high levels of sodium ion and urea by accumulating compatible osmolytes such as sorbitol, betaine, and myo-inositol. These osmolytes are involved in maintaining cell volume and electrolyte contents because they do not perturb the protein structure and function over a wide range of concentrations. Sorbitol is produced via the reduction of glucose by aldose reductase (AR), while betaine and myo-inositol are transported into the cells through specific transporters. Under hyperosmotic stress, transcriptions of genes encoding these proteins are highly induced. The induction of transcription was found to be mediated through the osmotic response elements (OREs) located in the 5' flanking sequences of these genes. We had earlier identified the OREs in human AR gene. In this study we purified and identified the osmotic response element binding protein (OREBP). OREBP is a transcription factor of approximately 200 kDa in size, characterized by a Rel-like DNA binding domain and a glutamine-rich transactivation domain. Dominant negative OREBP significantly diminished hyperosmotic AR gene induction. Immunohistochemical analysis showed that this transcription factor is rapidly translocated into the nucleus upon hyperosmotic stress.

BLNK: a central linker protein in B cell activation.

Linker or adapter proteins provide mechanisms by which receptors can amplify and regulate downstream effector proteins. We describe here the identification of a novel B cell linker protein, termed BLNK, that interfaces the B cell receptor-associated Syk tyrosine kinase with PLCgamma, the Vav guanine nucleotide exchange factor, and the Grb2 and Nck adapter proteins. Tyrosine phosphorylation of BLNK by Syk provides docking sites for these SH2-containing effector molecules that, in turn, permits the phosphorylation and/or activation of their respective signaling pathways. Hence, BLNK represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways and may regulate the biologic outcomes of B cell function and development.

Identification and cloning of centaurin-alpha. A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain.

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Mechanisms of thrombin receptor agonist specificity. Chimeric receptors and complementary mutations identify an agonist recognition site.

Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn87 in the Xenopus receptor's amino-terminal exodomain and Glu for Leu260 in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg5a in the human agonist peptide might normally interact with Glu260 in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu260 was converted to Arg. An "arginine scan" of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu260 region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu260 region and to a lesser degree Phe87 as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the "correct" agonist and inhibit responsiveness to an "incorrect" agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.

Primary structure of the soluble lactose binding lectin L-29 from rat and dog and interaction of its non-collagenous proline-, glycine-, tyrosine-rich sequence with bacterial and tissue collagenase.

A lactose-binding lectin from rat lung (RL-29) and a related lectin from Madin-Darby canine kidney (MDCK) cells have been analyzed with the primary goal of identifying post-translational modifications. The sequences show that RL-29 and the dog lectin are homologues of a lectin designated here as L-29 and elsewhere as CBP-35, epsilon BP, Mac-2, or L-34. RL-29 has a 140-amino-acid COOH-terminal carbohydrate-binding domain, a 20-amino-acid NH2-terminal domain, and an intervening domain consisting of 11 repeating elements rich in Pro, Gly, and Tyr (R-domain). The dog homologue has 14 repeating elements in its R-domain explaining its larger size. The sensitivity of the R-domain to bacterial collagenase allowed us to isolate the NH2-terminal domain and show that the NH2 terminus was blocked by acetylation and, in the accompanying paper (Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H., and Leffler, H. (1993) J. Biol. Chem. 268, 26712-26718), that the NH2-terminal domain is phosphorylated. In addition, we unexpectedly found an endogenous component, resembling 92-kDa type IV collagenase, that co-purified with L-29 and slowly digested the R-domain. Hence, L-29 is a substrate for bacterial and tissue collagenases even though the R-domain is non-collagenous. Moreover, the co-purification suggests a non-enzymatic interaction between 92-kDa collagenase and L-29.