Ralf Schülein

Disease‐causing V2 Vasopressin Receptors are Retained in Different Compartments of the Early Secretory Pathway

The G protein‐coupled V2 vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V2 vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease‐causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V2 vasopressin receptor mutants. We show that mutants L62P, ΔL62‐R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease‐causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.

Folding and cell surface expression of the vasopressin V2 receptor: requirement of the intracellular C‐terminus

We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C‐terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild‐type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin‐sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c‐myc epitope‐ or green fluorescent protein‐tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C‐terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length‐dependent reduction of cell surface expression. Replacement of positively charged residues within the C‐terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C‐terminus replaced by that of the β2‐adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N‐terminal part of the C‐terminus are necessary for correct folding and that C‐terminal residues are important for efficient cell surface expression.

Primary and secondary thyroid hormone transporters

Thyroid hormones (TH) are essential for the development of the human brain, growth and cellular metabolism. Investigation of TH transporters became one of the emerging fields in thyroid research after the discovery of inactivating mutations in the Monocarboxylate transporter 8 (MCT8), which was found to be highly specific for TH transport. However, additional transmembrane transporters are also very important for TH uptake and efflux in different cell types. They transport TH as secondary substrates and include the aromatic amino acid transporting MCT10, the organic anion transporting polypeptides (e.g. OATP1C1, OATP1A2, OPTP1A4) and the large neutral amino acid transporters (LAT1 and LAT2). These TH transporters characteristically possess 12 transmembrane spanners but due to the strong differing sequences between the three transporter families we assume an identical conformation is not very likely. In contrast to the others, the LAT family members form a heterodimer with the escort protein 4F2hc/CD98. A comparison of sequence proportions, locations and types of functional sensitive features for TH transport discovered by mutations, revealed that transport sensitive charged residues occur as conserved amino acids only within each family of the transporter types but not in all putative TH transporters. Based on the lack of highly conserved sensitive charged residues throughout the three transporter families as a common counterpart for the amino acid moiety of the substrates, we conclude that the molecular transport mechanism is likely organized either a) by different molecular determinants in the divergent transporter types or b) the counterparts for the substrates` amino acid moiety at the transporter are not any charged side chains but other proton acceptors or donators. However, positions of transport sensitive residues coincide at transmembrane helix 8 in the TH transporter MCT8, OATP1C1 and another amino acid transporter, the L-cystine and L-glutamate exchanger xCT, which is highly homologous to LAT1 and LAT2. Here we review the data available and compare similarities and differences between these primary and secondary TH transporters regarding sequences, topology, potential structures, trafficking to the plasma membrane, molecular features and locations of transport sensitive functionalities. Thereby, we focus on TH transporters occurring in the blood-brain barrier.

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.

Functional Rescue of the Nephrogenic Diabetes Insipidus-causing Vasopressin V2 Receptor Mutants G185C and R202C by a Second Site Suppressor Mutation

Mutations in the gene of the G protein-coupled vasopressin V2 receptor (V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the receptor introduce additional cysteine residues. Several groups have proposed that these residues might disrupt the conserved disulfide bond of the V2 receptor. To test this hypothesis, we first calculated a structure model of the extracellular receptor domains. The model suggests that the additional cysteine residues may form a second disulfide bond with the free, nonconserved extracellular cysteine residue Cys-195 rather than impairing the conserved bond. To address this question experimentally, we used the NDI-causing mutant receptors G185C and R202C. Their Cys-195 residues were replaced by alanine to eliminate the hypothetical second disulfide bonds. This second site mutation led to functional rescue of both NDI-causing mutant receptors, strongly suggesting that the second disulfide bonds are indeed formed. Furthermore we show that residue Cys-195, which is sensitive to “additional cysteine” mutations, is not conserved among the V2 receptors of other species and that the presence of an uneven number of extracellular cysteine residues, as in the human V2 receptor, is rare among class I G protein-coupled receptors.

A Single Negatively Charged Residue Affects the Orientation of a Membrane Protein in the Inner Membrane of Escherichia coliOnly When It Is Located Adjacent to a Transmembrane Domain

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane ofEscherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein (“positive inside” rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V2 receptor and the first cytoplasmic loop of the β2-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (Nin-Cout orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.

The hydrophobic amino acid residues in the membrane-proximal C tail of the G protein-coupled vasopressin V2 receptor are necessary for transport-competent receptor folding

It is believed that the membrane‐proximal C tail of the G protein‐coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L339, L340) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non‐polar side of this helix (F328, V332 and L336) are also transport‐relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S330, S331, S333, S334, S338) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport‐competent folding state in the early secretory pathway.

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.