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PubMed1995

A novel endoprotease responsible for the specific cleavage of transducin gamma subunit.

Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.

Amino Acid Sequence↗
PubMed1989

Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin).

The mechanism of activation of cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G alpha GTP (alpha subunit of the G-protein with GTP bound) with high affinity (100 +/- 50 nM) partially activates the enzyme (Vmax1 approxmately 0.05 Vmax to 0.10V max to trypsin-activated phosphodiesterase); binding of a second G alpha GTP with lower affinity (600 +/- 100 nM) induces maximal activation (Vmax2 approximately Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP-gamma S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.

As sensors for structure at the cytoplasmic face of rhodopsin, single-cysteine substitution mutants have been previously studied in the regions connecting helices III and IV and helices V and VI. In this paper we report on single-cysteine substitution mutants at amino acid positions 306-321, comprising the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with 11-cis-retinal all formed the characteristic rhodopsin chromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4, 4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 showed no reaction with 4-PDS. The mutants on illumination also showed wide variations in activating GT. The mutant Y306C showed almost no GT activation, I307C and N310C were poor, and the activity of the mutants M309C, F313C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate in the interaction with GT.

Amino Acid Sequence↗
PubMed2000

Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism.

Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, G(t), we have used kinetic light scattering, backed by direct biochemical assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the G(t)GDP substrate; (ii) to titrate G(t)GDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of K(m) = 2200 G(t)/microm(2) for G(t)GDP and K(m) = 230 microm for GTP; dissociation constants are K(d) = 530 G(t)/microm(2) for R*-G(t)GDP dissociation and K(d) = 270 microm for GDP release from R*G(t)GDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 G(t)/s at 22 degrees C and 1300 G(t)/s at 34 degrees C. The analysis provides a tool to allocate and quantify better the effects of chemical or mutational protein modifications to individual steps in signal transduction.

Animals↗
PubMed2001

Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB.

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

3T3 Cells↗
PubMed2006

Signal transfer from GPCRs to G proteins: role of the G alpha N-terminal region in rhodopsin-transducin coupling.

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).

Amino Acid Sequence↗
PubMed1990

Rhodopsin mutants that bind but fail to activate transducin.

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

Amino Acid Sequence↗
PubMed1999

TBL2, a novel transducin family member in the WBS deletion: characterization of the complete sequence, genomic structure, transcriptional variants and the mouse ortholog.

Williams-Beuren syndrome (WBS) is a developmental disorder with multi-system manifestations caused by haploinsufficiency for contiguous genes deleted in chromosome region 7q11.23. The size of the deletion is similar in most patients due to a genomic duplication that predisposes to unequal meiotic crossover events. While hemizygosity at the elastin locus is responsible for the cardiovascular features, the contribution of other genes to the WBS phenotype remains to be demonstrated. We have identified a novel gene, TBL2, in the common WBS deletion. TBL2 is expressed as a 2. 4-kb transcript predominantly in testis, skeletal muscle, heart and some endocrine tissues, with a larger approximately 5-kb transcript detected ubiquitously at lower levels. TBL2 encodes a protein with four putative WD40-repeats. An alternatively spliced transcript in TBL2 introduces a novel second exon with an in frame stop codon. This mRNA encodes a 75 amino acid protein with 43 amino acids identical to TBL2 at the N-terminus and no known functional domain. The mouse homolog, Tbl2, shows 84% sequence identity at the nucleotide level and 92% similarity at the amino acid level. Comparison of the mouse and human sequences identifies a conserved region that extends upstream of the previously published sequence with an initiation codon common to both species that adds 21 amino acids at the N-terminus. The Tbl2 gene has been mapped to mouse chromosome 5 in a region of conserved synteny with human 7q11.23. Since haploinsufficiency has been shown for other WD-repeat containing proteins, hemizygosity of TBL2 may contribute to some of the aspects of the complex WBS phenotype.

Amino Acid Sequence↗