The sequence after the signal peptide of the G protein-coupled endothelin B receptor is required for efficient translocon gating at the endoplasmic reticulum membrane.

The heptahelical G protein-coupled receptors (GPCRs) must reach their correct subcellular location to exert their function. Receptor domains relevant for receptor trafficking include signal sequences mediating receptor integration into the membrane of the endoplasmic reticulum (ER) and anterograde or retrograde transport signals promoting receptor sorting into the vesicles of the secretory pathway. In addition, receptors must be correctly folded to pass the quality control system of the early secretory pathway. Taking the endothelin B receptor as a model, we describe a new type of a transport-relevant GPCR domain. Deletion of this domain (residues Glu(28) to Trp(54)) leads to a fully functional receptor protein that is expressed at a lower level than the wild-type receptor. Subcellular localization experiments and glycosylation state analyses demonstrate that the mutant receptor is neither misfolded, retained intracellularly, nor misrouted. Fluorescence recovery after photobleaching analyses demonstrate that constitutive internalization is also not affected. By using an in vitro prion protein targeting assay, we show that this domain is necessary for efficient translocon gating at the ER membrane during early receptor biogenesis. Taken together, we identified a novel transport-relevant domain in the GPCR protein family. Our data may also be relevant for other GPCRs and unrelated integral membrane proteins.

Local RNA target structure influences siRNA efficacy: a systematic global analysis.

The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.

The signal peptide of the rat corticotropin-releasing factor receptor 1 promotes receptor expression but is not essential for establishing a functional receptor.

Approximately 5-10% of the GPCRs (G-protein-coupled receptors) contain N-terminal signal peptides that are cleaved off during receptor insertion into the ER (endoplasmic reticulum) membrane by the signal peptidases of the ER. The reason as to why only a subset of GPCRs requires these additional signal peptides is not known. We have recently shown that the signal peptide of the human ET(B)-R (endothelin B receptor) does not influence receptor expression but is necessary for the translocation of the receptor's N-tail across the ER membrane and thus for the establishment of a functional receptor [Köchl, Alken, Rutz, Krause, Oksche, Rosenthal and Schülein (2002) J. Biol. Chem. 277, 16131-16138]. In the present study, we show that the signal peptide of the rat CRF-R1 (corticotropin-releasing factor receptor 1) has a different function: a mutant of the CRF-R1 lacking the signal peptide was functional and displayed wild-type properties with respect to ligand binding and activation of adenylate cyclase. However, immunoblot analysis and confocal laser scanning microscopy revealed that the mutant receptor was expressed at 10-fold lower levels than the wild-type receptor. Northern-blot and in vitro transcription translation analyses precluded the possibility that the reduced receptor expression is due to decreased transcription or translation levels. Thus the signal peptide of the CRF-R1 promotes an early step of receptor biogenesis, such as targeting of the nascent chain to the ER membrane and/or the gating of the protein-conducting translocon of the ER membrane.

The corticotropin-releasing factor receptor type 2a contains an N-terminal pseudo signal peptide.

The corticotropin-releasing factor receptor type 2a (CRF(2(a)) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(a)) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn(13) by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially.

The signal peptide of the G protein-coupled human endothelin B receptor is necessary for translocation of the N-terminal tail across the endoplasmic reticulum membrane.

The initial step of the intracellular transport of G protein-coupled receptors, their insertion into the membrane of the endoplasmic reticulum, follows one of two different pathways. Whereas one group uses the first transmembrane domain of the mature receptor as an uncleaved signal anchor sequence for this process, a second group possesses additional cleavable signal peptides. The reason this second subset requires the additional signal peptide is not known. Here we have assessed the functional significance of the signal peptide of the endothelin B (ET(B)) receptor in transiently transfected COS.M6 cells. A green fluorescent protein-tagged ET(B) receptor mutant lacking the signal peptide was nonfunctional and retained in the endoplasmic reticulum, suggesting that it has a folding defect. To determine the defect in more detail, ET(B) receptor fragments containing the N-terminal tail, first transmembrane domain, and first cytoplasmic loop were constructed. We assessed N tail translocation across the endoplasmic reticulum membrane in the presence and absence of a signal peptide and show that the signal peptide is necessary for N tail translocation. We postulate that signal peptides are necessary for those G protein-coupled receptors for which post-translational translocation of the N terminus is impaired or blocked by the presence of stably folded domains.