The corticotropin-releasing factor receptor type 2a (CRF 2(a) R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF 1 R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF 2(a) R in comparison with the CRF 1 R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G s protein, G i -mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF 1 R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.The family of corticotropin-releasing factor (CRF) 3 receptors encompasses two subtypes, the CRF 1 R and CRF 2 R (1, 2). The CRF 1 R is expressed mainly in the anterior pituitary and plays a central role in the regulation of the hypothalamic-pituitaryadrenal stress axis in mammals (3). It binds CRF with high affinity and mediates ACTH release from the pituitary leading to cortisol biosynthesis in the adrenal cortex. A large body of evidence points to a major role of the receptor in mediating CRF effects in anxiety and depressive disorders (4 -6).In the case of the CRF 2 R, three splice variants have been described as follows: the CRF 2(a) R, CRF 2(b) R, and CRF 2(c) R. All splice variants bind CRF with low affinity and the urocortins 1-3 with high affinity. They are involved in the regulation of feeding behavior (7) and in recovery from a stress response (8).It is likely that they are also involved in modulating anxietyrelated behavior.Both the CRF 1 R and the CRF 2(a) R usually couple to the G s / adenylyl cyclase system and consequently increase cytosolic cAMP as a second messenger. However, a promiscuous coupling behavior was described previously in particular for the CRF
The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.
The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher-Nadel, C., and Lindley, I. J.
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