Daniel F. Ortiz

Tyrosine-kinase-dependent recruitment of RGS12 to the N-type calcium channel.

γ-Aminobutyric acid (GABA)B receptors couple to G o to inhibit N-type calcium channels in embryonic chick dorsal root ganglion neurons. The voltage-independent inhibition, mediated by means of a tyrosine-kinase pathway, is transient and lasts up to 100 seconds. Inhibition of endogenous RGS12, a member of the family of regulators of G-protein signalling, selectively alters the time course of voltage-independent inhibition. The RGS12 protein, in addition to the RGS domain, contains PDZ and PTB domains. Fusion proteins containing the PTB domain of RGS12 alter the rate of termination of the GABAB signal, whereas the PDZ or RGS domains of RGS12 have no observable effects. Using primary dorsal root ganglion neurons in culture, here we show an endogenous agonist-induced tyrosine-kinase-dependent complex of RGS12 and the calcium channel. These results indicate that RGS12 is a multifunctional protein capable of direct interactions through its PTB domain with the tyrosine-phosphorylated calcium channel. Recruitment of RGS proteins to G-protein effectors may represent an additional mechanism for signal termination in G-protein-coupled pathways.

A Yeast ATP-binding Cassette-type Protein Mediating ATP-dependent Bile Acid Transport

ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases. Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiaealso exhibit ATP-dependent bile acid transport. ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a K m of 63 μmfor taurocholate. The BAT1 (bileacid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins. ATP-dependent bile acid transport was abolished when theBAT1 coding region was deleted from the genome and restored upon reintroduction of the gene. The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function. Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

Regulators of G Protein Signaling Proteins as Determinants of the Rate of Desensitization of Presynaptic Calcium Channels

Norepinephrine inhibits ω-conotoxin GVIA-sensitive presynaptic Ca2+ channels in chick dorsal root ganglion neurons through two pathways, one mediated by Go and the other by Gi. These pathways desensitize at different rates. We have found that recombinant Gα interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of Go- and Gi-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against Gα interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with Gi and Go selectively, giving rise to distinct time courses of transmitter-mediated effects.

MRP3, a new ATP-binding cassette protein localized to the canalicular domain of the hepatocyte

Bile secretion in liver is driven in large part by ATP-binding cassette (ABC)-type proteins that reside in the canalicular membrane and effect ATP-dependent transport of bile acids, phospholipids, and non-bile acid organic anions. Canalicular ABC-type proteins can be classified into two subfamilies based on membrane topology and sequence identity: MDR1, MDR3, and SPGP resemble the multidrug resistance (MDR) P-glycoprotein, whereas MRP2 is similar in structure and sequence to the multidrug resistance protein MRP1 and transports similar substrates. We now report the isolation of the rMRP3 gene from rat liver, which codes for a protein 1522 amino acids in length that exhibits extensive sequence similarity with MRP1 and MRP2. Northern blot analyses indicate that rMRP3 is expressed in lung and intestine of Sprague-Dawley rats as well as in liver of Eisai hyperbilirubinemic rats and TR- mutant rats, which are deficient in MRP2 expression. rMRP3 expression is also transiently induced in liver shortly after birth and during obstructive cholestasis. Antibodies raised against MRP3 recognize a polypeptide of 190-200 kDa, which is reduced in size to 155-165 kDa after treatment with endoglycosidases. Immunoblot analysis and immunoconfocal microscopy indicate that rMRP3 is present in the canalicular membrane, suggesting that it may play a role in bile formation.

Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.