Figure 7 - uploaded by Zdenka Drastichova
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Effect of TRH concentration on dissociation/association of G q/11 a complexes HEK293 cells were treated with TRH at different concentrations (10, 1, 0.1, 0.01, and 0.001 mM) for 16 h. Samples of the postnuclear supernatants from the control and TRH-treated cells were solubilized with Brij 56 (A) or LM (B) and separated on 6% – 15% linear gradient non-denaturating polyacrylamide gels. The resolved complexes were identified after electrotransfer onto PVDF membranes and incubation with anti-G q/11 a antibody.
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Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors. At least some types of G-protein α subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists. However, the mechanism underlyi...
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... would also be detectable with lower concentrations of the hormone. Therefore HEK293 cells were treated for 16 h with TRH in the 10 25 -10 29 M range. The PNS from the control and TRH-treated cells were then solubilized by LM or Brij 56, protein complexes separated by CN-PAGE and G q/11 a proteins were detected by using specific antibodies (Fig. 7). Whereas dissociation of the G q/11 protein complex in the 140 kDa region was more pronounced in samples of cells treated with higher concentrations of TRH (10 25 -10 27 M) and it was not observed in the case of 1Â10 29 M TRH, the decomposition of the 300 kDa G q/11 protein complex was detected only in the presence of 1Â10 25 M TRH. ...
Citations
... Subsequently, Petrou and Tashjian (1998) investigated the hypothesis that G q α and G 11 α need not dissociate from receptors during internalization at the plasma membrane, and they confirmed that TRH-R and the G 11 α subunit are internalized together in clathrin-coated vesicles in GH 4 C 1 cells. Later studies based on the use of clear native polyacrylamide gel electrophoresis revealed the presence of preassembled complexes of rat TRH-R1 and G q/11 α (Drastichova and Novotny, 2012a). Treatment with TRH resulted in the dissociation of high-molecular-weight complexes of G q/11 α, the formation of low-molecular-weight complexes containing G q/11 α, and the concomitant translocation of this protein to the cytosol (Drastichova and Novotny, 2012b). ...
Thyrotropin-releasing hormone (TRH) is an important endocrine agent that regulates the function of cells in the anterior pituitary and the central and peripheral nervous systems. By controlling the synthesis and release of thyroid hormones, TRH affects many physiological functions, including energy homeostasis. This hormone exerts its effects through G protein-coupled TRH receptors, which signal primarily through Gq/11 but may also utilize other G protein classes under certain conditions. Because of the potential therapeutic benefit, considerable attention has been devoted to the synthesis of new TRH analogs that may have some advantageous properties compared with TRH. In this context, it may be interesting to consider the phenomenon of biased agonism and signaling at the TRH receptor. This possibility is supported by some recent findings. Although knowledge about the mechanisms of TRH receptor-mediated signaling has increased steadily over the past decades, there are still many unanswered questions, particularly about the molecular details of post-receptor signaling. In this review, we summarize what has been learned to date about TRH receptor-mediated signaling, including some previously undiscussed information, and point to future directions in TRH research that may offer new insights into the molecular mechanisms of TRH receptor-triggered actions and possible ways to modulate TRH receptor-mediated signaling.
... The lower band that is recognized by the antibody is Gnαq (40 kDA; accession #: AAH11169.1) [39], which shares an 82% identity with Gnα11 (accession #: AAB36839.1) in the area of the epitope chosen by the producer of the antibody (amino acids 3-39 within the N-terminus). Under the denaturing conditions used for western blotting this antibody recognizes the similar epitopes of Gnα11 and Gnαq (Fig 9C), but under the conditions of the immunofluorescence experiments ( Fig 9B) the antibody displays a higher specificity for the original target Gnα11, and cross-reactivity with similar epitopes remains at a minimum. ...
Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 μM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gnα11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gnα11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility.
Mammalian type Opsin 5 (Opn5m), a UV-sensitive GPCR opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-knockout cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by co-transfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C-terminus, in the preferential activation of Gα14 by Opn5m. Fluorescent in situ hybridization revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types.
Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP1 production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15–31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM15–52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.
