Claudia Rutz

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

The corticotropin-releasing factor receptor type 2a (CRF2(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 CRF1 receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF2(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 Asn13 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 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 receptors 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.

Membrane Targeting and Determination of Transmembrane Topology of the Human Vasopressin V2 Receptor

The human vasopressin V2 receptor belongs to the large family of G-protein-coupled receptors, which possess seven transmembrane helices, an extracellular N terminus and an intracellular C terminus. We have determined the sequence requirements of the V2 receptor for membrane insertion and correct topology for the inner membrane of Escherichia coli with the PhoA/LacZ gene fusion system. In addition, we have studied the signals for its membrane insertion and correct topology for the membrane of the endoplasmic reticulum of the authentic eucaryotic transport system. To this end, we have extended the PhoA/LacZ gene fusion system for the first time to eucaryotic cells, i.e. transiently transfected COS.M6 cells. Truncated V2 receptor sequences were fused to PhoA and LacZ and expressed in both E. coli and COS.M6 cells. Cells were fractionated, and LacZ/PhoA activity assays and immunoblots were performed. We show here that a V2 receptor fragment consisting of the N terminus, the first transmembrane segment and the first cytoplasmic loop (71 amino acids) provided sufficient information for membrane insertion and correct orientation (extracellular N terminus) in both procaryotic and eucaryotic cells. Our data differ substantially from those obtained for the human β2-adrenergic receptor expressed in E. coli (Lacatena, R. M., Cellini, A., Scavizzi, F., and Tocchini-Valentini, G. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10521-10525). To establish correct topology, the β2-adrenergic receptor requires a larger receptor portion, including the three N-terminal transmembrane segments and/or parts of the second cytoplasmic loop. The present data show that the observations made for the β2-adrenergic receptor cannot be applied to G-protein-coupled receptors generally.

Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor

The thyrotropin receptor [thyroidstimulating hormone receptor (TSHR)], a G‐proteincoupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low‐molecular‐weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptors transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site‐directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling‐sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.—Kleinau, G., Haas, A.‐K., Neumann, S., Worth, C. L., Hoyer, I., Furkert, J., Rutz, Gershengorn, M. C, Schülein, R., Krause, G. Signalingsensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor. FASEBJ. 24, 2347–2354 (2010). www.fasebj.org

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity

The corticotropin-releasing factor receptor type 2a (CRF2(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 (CRF1R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF2(a)R in comparison with the CRF1R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the Gs protein, Gi-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF1R 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.

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-5935.50) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

The pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization

Background: The corticotropin-releasing factor receptor type 2a is a GPCR possessing an N-terminal pseudo signal peptide with unknown function. Results: The pseudo signal peptide prevents receptor oligomerization. Conclusion: We have identified a monomeric GPCR and a novel functional domain playing a role in receptor oligomerization. Significance: The pseudo signal peptide may be useful to study the functional significance of GPCR oligomerization in general. N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF2(a)R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF2(a)R and the homologous corticotropin-releasing factor receptor type 1 (CRF1R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF2(a)R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

Background: GPCRs may be expressed in a monomer/dimer equilibrium at the plasma membrane. Results: For the dimeric CRF1R, we found a constant monomer/dimer equilibrium not only at the plasma membrane but also in the ER. Conclusion: The monomer/dimer equilibrium of the CRF1R is established already in the ER. Significance: Our findings shed new light on the monomer/dimer equilibrium of GPCRs and on ER functions. G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment.

Mutations that silence constitutive signaling activity in the allosteric ligand-binding site of the thyrotropin receptor

The thyrotropin receptor (TSHR) exhibits elevated cAMP signaling in the basal state and becomes fully activated by thyrotropin. Previously we presented evidence that small-molecule ligands act allosterically within the transmembrane region in contrast to the orthosteric extracellular hormone-binding sites. Our goal in this study was to identify positions that surround the allosteric pocket and that are sensitive for inactivation of TSHR. Homology modeling combined with site-directed mutagenesis and functional characterization revealed seven mutants located in the allosteric binding site that led to a decrease of basal cAMP signaling activity. The majority of these silencing mutations, which constrain the TSHR in an inactive conformation, are found in two clusters when mapped onto the 3D structural model. We suggest that the amino acid positions identified herein are indicating locations where small-molecule antagonists, both neutral antagonists and inverse agonists, might interfere with active TSHR conformations.

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 Glu28 to Trp54) 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.