Pertussis toxin-sensitive signaling of melanocortin-4 receptors in hypothalamic GT1-7 cells defines agouti-related protein as a biased agonist.

Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G(s) proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G(s). However, some mutants displayed enhanced G(s) coupling, suggesting that signaling pathways independent of G(s) may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G(s) signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G(i/o) proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G(s) and G(i/o) proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G(i/o) signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G(s) activation not only by competitive antagonism but additionally by initiating G(i/o) protein-induced signaling as a biased agonist.

Role of Chemosensory TRP Channels in Lung Cancer.

Transient receptor potential (TRP) channels represent a large family of cation channels and many members of the TRP family have been shown to act as polymodal receptor molecules for irritative or potentially harmful substances. These chemosensory TRP channels have been extensively characterized in primary sensory and neuronal cells. However, in recent years the functional expression of these proteins in non-neuronal cells, e.g., in the epithelial lining of the respiratory tract has been confirmed. Notably, these proteins have also been described in a number of cancer types. As sensor molecules for noxious compounds, chemosensory TRP channels are involved in cell defense mechanisms and influence cell survival following exposure to toxic substances via the modulation of apoptotic signaling. Of note, a number of cytostatic drugs or drug metabolites can activate these TRP channels, which could affect the therapeutic efficacy of these cytostatics. Moreover, toxic inhalational substances with potential involvement in lung carcinogenesis are well established TRP activators. In this review, we present a synopsis of data on the expression of chemosensory TRP channels in lung cancer cells and describe TRP agonists and TRP-dependent signaling pathways with potential relevance to tumor biology. Furthermore, we discuss a possible role of TRP channels in the non-genomic, tumor-promoting effects of inhalational carcinogens such as cigarette smoke.

TSH induces metallothionein 1 in thyrocytes via Gq/11- and PKC-dependent signaling.

Metallothioneins (MTs) are cytoprotective proteins acting as scavengers of toxic metal ions or reactive oxygen species. MTs are upregulated in follicular thyroid carcinoma and are regarded as a marker of thyroid stress in Graves' disease. However, the mechanism of MT regulation in thyrocytes is still elusive. In other cellular systems, cAMP-, calcium-, or protein kinase C (PKC)-dependent signaling cascades have been shown to induce MT expression. Of note, all of these three pathways are activated following the stimulation of the TSH receptor (TSHR). Thus, we hypothesized that TSH represents a key regulator of MT expression in thyrocytes. In fact, TSHR stimulation induced expression of MT isoform 1X (MT1X) in human follicular carcinoma cells. In these cells, Induction of MT1X expression critically relied on intact Gq/11 signaling of the TSHR and was blocked by chelation of intracellular calcium and inhibition of PKC. TSHR-independent stimulation of cAMP formation by treating cells with forskolin also led to an upregulation of MT1X, which was completely dependent on PKA. However, inhibition of PKA did not affect the regulation of MT1X by TSH. As in follicular thyroid carcinoma cells, TSH also induced MT1 protein in primary human thyrocytes, which was PKC dependent as well. In summary, these findings indicate that TSH stimulation induces MT1X expression via Gq/11 and PKC, whereas cAMP-PKA signaling does not play a predominant role. To date, little has been known regarding cAMP-independent effects of TSHR signaling. Our findings extend the knowledge about the PKC-mediated functions of the TSHR.

Alternative G protein coupling and biased agonism: new insights into melanocortin-4 receptor signalling.

The melanocortin-4 receptor (MC4R) is a prototypical G protein-coupled receptor (GPCR) that plays a considerable role in controlling appetite and energy homeostasis. Signalling initiated by MC4R is orchestrated by multiple agonists, inverse agonism and by interactions with accessory proteins. The exact molecular events translating MC4R signalling into its physiological role, however, are not fully understood. This review is an attempt to summarize new aspects of MC4R signalling in the context of its recently discovered alternative G protein coupling, and to give a perspective on how future research could improve our knowledge about the intertwining molecular mechanisms that are responsible for the regulation of energy homeostasis by the melanocortin system.

G13-dependent activation of MAPK by thyrotropin.

Stimulation of the thyrotropin receptor (TSHR) activates G proteins of all four subfamilies (G(s), G(i/o), G(q/11), and G(12/13)). Whereas G(s)/cAMP-dependent cellular responses upon TSHR stimulation are well established, other signaling pathways are less characterized. We evaluated TSH-elicited cellular responses in human follicular thyroid carcinoma cells stably expressing the TSHR and in primary, nonneoplastic human thyrocytes. In these cellular models, stimulation with TSH caused activation of p44/42 MAPK and subsequent induction of c-Fos. MAPK stimulation occurred independently of G(s), G(i/o), and G(q/11) signaling. Dominant negative constructs of G(12) or G(13) as well as shRNA-mediated suppression of Galpha(12) or Galpha(13) revealed that MAPK activation was dependent on G(13) but not on G(12) signaling. Furthermore, G(13)-dependent transactivation of the epidermal growth factor receptor was necessary for MAPK activation in follicular carcinoma cells, whereas EGFR was not involved in MAPK activation in nonneoplastic primary thyrocytes. The use of bacterial inhibitors of monomeric GTPases revealed that MAPK activation proceeded independently of Rho proteins but was clostridial toxin B-sensitive, suggesting involvement of Cdc42 or Rac. Thus, our data shed new light on cAMP-independent TSHR signaling and identify the first G(13)-dependent TSHR signaling pathway in human thyrocytes.