Thrombin-induced Ca2+ mobilization in vascular smooth muscle utilizes a slowly ribosylating pertussis toxin-sensitive G protein: evidence for the involvement of a G protein in inositol trisphosphate-dependent Ca2+ release

The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the Gα(i2), Gα(i3), Gα(s), Gβ, and Gβ protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca mobilization, we investigated the effect of PT on Ca responses using fluorescence spectroscopy and the Ca indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 μg/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca response was inhibited by approximately 72%, suggesting that the major fraction of the Ca response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the β-subunit of G proteins because quantitative immunoblots showed that levels of the β-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca pool because Ca release induced by agents that release Ca directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [H]inositol trisphosphate (IP) formation because the level of inhibition of IP formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT- sensitive species is the major G protein pathway that couples thrombin- receptor activation to Ca mobilization. This G protein appears to be involved not in the mechanisms that generate IP but rather possibly in coupling at the level of the intracellular Ca store

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat-shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data