Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase.

Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size.     

Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.     

Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate.

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.     

How a G protein binds a membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.     

A virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses

During ongoing C-type retrovirus infection, the probability of leukemia caused by insertional gene activation is markedly increased by the emergence of recombinant retroviruses that repeatedly infect host cells. The murine mink cell focus-inducing (MCF) viruses with this property have acquired characteristic changes in the N-terminal domain of their envelope glycoprotein that specify binding to a different receptor than the parental ecotropic virus. In this report, we show that MCF virus infection occurs through binding to this receptor (termed Syg1) and, remarkably, by a second mechanism that does not utilize the Syg1 receptor. By the latter route, the N-terminal domain of the ecotropic virus glycoprotein expressed on the cell surface in a complex with its receptor activates the fusion mechanism of the MCF virus in trans. The rate of MCF virus spread through a population of permissive human cells was increased by establishment of trans activation, indicating that Syg1 receptor-dependent and -independent pathways function in parallel. Also, trans activation shortened the interval between initial infection and onset of cell-cell fusion associated with repeated infection of the same cell. Our findings indicate that pathogenic retrovirus infection may be initiated by virus binding to cell receptors or to the virus envelope glycoprotein of other viruses expressed on the cell surface. Also, they support a broader principle: that cooperative virus-virus interactions, as well as virus-host interactions, shape the composition and properties of the retrovirus quasispecies.     

A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E

Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.     

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.