K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.     

Comparison by photoaffinity labeling of the proteins involved in GTP hydrolysis from 70S ribosomes and a 5S RNA-protein complex from Bacillus stearothermophilus.

Photoaffinity labeling of 70S ribosomes from B. stearothermophilus by [³H]-1-(4-azidophenyl)-2-(5′-guanyl) pyrophosphate (APh-GDP) in the presence of fusidate and elongation factor G (EF-G) results in incorporation of tritium in the 50S proteins BL2, BL10 and BL22. Irradiation of the corresponding 5S RNA-protein complex in the presence of the GDP derivative gives only incorporation of tritium in BL10 and BL22. The proteins BL10 and BL22 comigrate in two dimensional gel electrophoresis with the 50S ribosomal proteins EL11 and EL18 from E. coli. The result suggests that the region at or near the guanine nucleotide binding site of the ribosome and the complex are the same. Since previous work has shown that the latter two are labeled upon irradiation of the ribosome with [³H]-APh-GDP, it is concluded that ribosomes from E. coli and B. stearothermophilus have structurally related GTPase sites.     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.