Ricardo Hermosilla

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.

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.

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.

Rescue of a Nephrogenic Diabetes Insipidus-causing Vasopressin V2 Receptor Mutant by Cell-penetrating Peptides

Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V2 receptor (V2R) leading to nephrogenic diabetes insipidus. Transport-defective V2Rs fall into two classes: those retained exclusively in the endoplasmic reticulum (ER) and those reaching post-ER compartments such as the ER/Golgi intermediate compartment. Although numerous chemical or pharmacological chaperones that rescue the transport of ER-retained membrane proteins are known, substances acting specifically in post-ER compartments have not been described as yet. Using the L62P (ER-retained) and Y205C (reaching post-ER compartments) mutants of the V2R as a model, we show here that the cell-penetrating peptide penetratin and its synthetic analog KLAL rescue the transport of the Y205C mutant. In contrast, the location of the L62P mutant is not influenced by either peptide because the peptides are unable to enter the ER. We also show data indicating that the peptide-mediated transport rescue is associated with an increase in cytosolic Ca2+ concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments.

Polarized cell surface expression of the green fluorescent protein‐tagged vasopressin V2 receptor in Madin Darby canine kidney cells

We have analyzed the polarized cell surface expression of the G protein‐coupled vasopressin V2 receptor (V2 receptor) in Madin‐Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)‐tagged receptors. Cell surface biotinylation assays with stably transfected filter‐grown cells expressing alkaline phosphatase (PhoA)‐tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter‐ and glass‐grown MDCK cells stably transfected with a GFP‐tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP‐tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP‐tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein‐coupled receptors. The methodology may be applicable to any cell type, even to tissue‐derived, primary cultured cells; coinjection of transport‐regulating compounds should also be possible.

Derlin-1 and p97/Valosin-Containing Protein Mediate the Endoplasmic Reticulum-Associated Degradation of Human V2 Vasopressin Receptors

The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues.

The G protein‐coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein‐coupled receptors. Here we have assessed the properties of the V2 receptor after site‐directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non‐functional and located mostly in the cells interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X‐linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin‐stimulated cAMP formation with a 35‐fold increased EC50 value and with a reduced ECmax, indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI‐causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.

Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site

hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

A Ser/Thr cluster within the C‐terminal domain of the rat prostaglandin receptor EP3α is essential for agonist‐induced phosphorylation, desensitization and internalization

1 Two isoforms of the rat prostaglandin E2 receptor, rEP3α‐R and rEP3β‐R, differ only in their C‐terminal domain. To analyze the function of the rEP3‐R C‐terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C‐terminal domain of the rEP3α‐R was mutated to Ala and both isoforms and the receptor mutant (rEP3α‐ST341‐349A‐R) were stably expressed in HEK293 cells. 2 All rEP3‐R receptors showed a similar ligand‐binding profile. They were functionally coupled to Gi and reduced forskolin‐induced cAMP‐formation. 3 Repeated exposure of cells expressing the rEP3α‐R isoform to PGE2 reduced the agonist induced inhibition of forskolin‐stimulated cAMP‐formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi‐response as well as plasma membrane localization of the rEP3β‐R and the rEP3α‐ST341‐349A‐R were not affected by prior agonist‐stimulation. 4 Agonist‐stimulation of HEK293‐rEP3α‐R cells induced a time‐ and dose‐dependent phosphorylation of the receptor most likely by G protein‐coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist‐stimulation the rEP3β‐R was not phosphorylated and the rEP3α‐ST341‐349A‐R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist‐induced desensitization of the rEP3α‐R isoform is mediated most likely by a GRK‐dependent phosphorylation of Ser/Thr residues 341–349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization.

Corrigendum to: The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues: [FEBS Letters 466 (2000) 101-106]

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