No abstract

A facile method for isolating genes that encode interacting proteins has been developed with a polypeptide probe that contains an amino-terminal extension with recognition sites for a monoclonal antibody, a specific endopeptidase, and a site-specific protein kinase. This probe, containing the basic region-leucine zipper dimerization motif of c-Fos, was used to screen a complementary DNA library. A complementary DNA that encoded a member of the basic-helix-loop-helix-zipper (bHLH-Zip) family of proteins was isolated. The complementary DNA-encoded polypeptide FIP (Fos interacting protein) bound to oligonucleotide probes that contained DNA binding motifs for other HLH proteins. When cotransfected with c-Fos, FIP stimulated transcription of an AP-1-responsive promoter.

Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. Following the identification of the founding member of this family, dTRP, which is from a Drosophila mutant with abnormal visual signal transduction (2), mammalian homologues have been cloned and all of them contain a six-transmembrane domain followed by a TRP motif (XWKFXR). Based on homology, they are divided into three subfamilies: TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) (3). Members of the TRPM subfamily have unusually long cytoplasmic tails at both ends of the channel domain, and some of the family members have an enzyme domain in the C-terminal region. Despite their similarities of structure, TRPMs have different ion-conductive properties, activation mechanisms, and putative biological functions. TRPM1 is down-regulated in metastatic melanomas (4). TRPM2 is a Ca 2ϩ -permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (5, 6), and changes in redox status (7). The TRPM2 gene is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holosencephaly (8). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca 2ϩ -permeable channel (9); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca 2ϩ (10). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (11). TRPM7, another bifunctional protein, has kinase activity in addition to its ion channel activity. TRPM7 is regulated by Mg 2ϩ -ATP and/or inositol 1,4,5-disphosphate and is required for cell viability (12-14). TRPM8 is up-regulated in prostate cancer and other malignancies (15). Recently, it has been shown to be a receptor that senses cold stimuli (16,17).Using a bioinformatics approach, we have identified a member of the human TRPM subfamily that we have called hTRPM3, consistent with the unified TRP nomenclature (3). hTRPM3 contains long N and C termini, although it does not contain any additional enzymatic features. hTRPM3 mRNA is expressed primarily in kidney with lower levels in brain, testis, and spinal cord. When expressed in HEK 293 cells, hTRPM3 is co-localized with the plasma membrane and is capable of mediating Ca 2ϩ entry. This hTRPM3-mediated Ca 2ϩ conductance * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.Tie2 (also called Tek) is a member of a novel family of receptor tyrosine kinases (RTKs) (16,17,37,42,72) that are expressed predominantly on endothelial cells or their embryonic precursors (14,16,17,37,42) and that are required for normal vascular development (15,52,55). Functional disruption of Tie2 in transgenic mice results in embryonic lethality by day E9.5 to 10.5 with effects on the microvasculature resulting in reduced numbers of endothelial cells and abnormalities of vascular morphogenesis (15,55). Knockout of the activating Tie2 ligand, angiopoietin-1 (Ang1), or overexpression of a related, inhibitory ligand, angiopoietin-2 (Ang2), resulted in phenotypes similar to the Tie2 knockout (43, 64). Taken together, these findings suggest a role for Tie2 in endothelial cell maintenance, survival, and/or vascular morphogenesis (24).In addition to a role in embryonic vascular development, data from our laboratory suggest that Tie2 plays an important role in the adult vasculature. For example, Tie2 expression is increased in the vasculature of malignant breast tumors (49), and a soluble extracellular domain of Tie2 inhibits tumor angiogenesis and growth (39). Tie2 is also broadly expressed and tyrosine phosphorylated in a variety of adult tissues in which the endothelium is normally quiescent (69). These findings are consistent with a dual role for Tie2 in both the growth and the maintenance of the adult vasculature.To better understand the role of Tie2 in vascular growth and maintenance, it will be important to identify the signal transduction pathways responsible for these functions. Currently, little is known about the specific signaling proteins and pathways utilized by Tie2. We demonstrated previously that Tie2 a...

The clinical features of long QT syndrome result from episodic life-threatening cardiac arrhythmias, specifically the polymorphic ventricular tachycardia torsades de pointes. KVLQT1 has been established as the human chromosome 11-linked gene responsible for more than 50% of inherited long QT syndrome. Here we describe the cloning of a full-length KVLQT1 cDNA and its functional expression. KVLQT1 encodes a 676-amino acid polypeptide with structural characteristics similar to voltage-gated potassium channels. Expression of KvLQT1 in Xenopus oocytes and in human embryonic kidney cells elicits a rapidly activating, K ؉ -selective outward current. The I Kr -specific blockers, E-4031 and dofetilide, do not inhibit KvLQT1, whereas clofilium, a class III antiarrhythmic agent with the propensity to induce torsades de pointes, substantially inhibits the current. Elevation of cAMP levels in oocytes nearly doubles the amplitude of KvLQT1 currents. Coexpression of minK with KvLQT1 results in a conductance with pharmacological and biophysical properties more similar to I Ks than other known delayed rectifier K ؉ currents in the heart.