Structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors

Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.     

Variant amino acids in the extracellular loops of murine and human vasopressin V2 receptors account for differences in cell surface expression and ligand affinity

Cloning and sequencing of the murine chromosomal region XB harboring the murine vasopressin V(2) receptor (mV(2)R) gene and comparison with the orthologous human Xq28 region harboring the human vasopressin V(2) receptor (hV(2)R) revealed conservation of the genomic organization and a high degree of sequence identity in the V(2)R coding regions. Despite an identity of 87% of the amino acid sequences, both receptors show marked functional differences upon stable expression in Chinese hamster ovary cells: the mV(2)R displayed a 5-fold higher affinity for [(3)H]AVP than the human ortholog; similar differences were found for the AVP-mediated activation of adenylyl cyclase. Saturation binding experiments with transiently transfected intact COS.M6 cells showed that the mV(2)R was 3- to 5-fold less abundantly expressed at the cell surface than the hV(2)R. Laser scanning microscopy of fusion proteins consisting of the V(2)Rs and green fluorescent protein (GFP) (mV(2)R/GFP, hV(2)R/GFP) demonstrated that the hV(2)R/GFP was efficiently transported to the plasma membrane, whereas the mV(2)R/GFP was localized mainly within the endoplasmic reticulum. Chimeric hV(2)Rs, in which the first and/or second extracellular loop(s) were replaced by the corresponding loop(s) of the mV(2)R, revealed that the second extracellular loop accounts for the differences in ligand binding, but the first extracellular loop accounts for the reduced cell surface expression. The exchange of lysine 100 by aspartate in the first extracellular loop of hV(2)R was sufficient to reduce cell surface expression, which was accompanied by intracellular retention as observed in laser scanning microscopy analysis. Conversely, the exchange of aspartate 100 by lysine in the mV(2)R increased the cell surface expression and resulted in predominant plasma membrane localization. Thus, a single amino acid difference in the first extracellular loop between mV(2)R and hV(2)R determines the efficiency of cell surface expression.     

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 V(2) receptor (V(2)R) leading to nephrogenic diabetes insipidus. Transport-defective V(2)Rs 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 V(2)R 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 Ca(2+) concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments.     

Assembly and cell surface expression of KA-2 subunit-containing kainate receptors

Kainate receptors (KARs) modulate synaptic transmission at both pre-synaptic and post-synaptic sites. The overlap in the distribution of KA-2 and GluR6/7 subunits in several brain regions suggests the co-assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5-7, the KA-2 subunit is unable to form functional homomeric KAR channels. We expressed the KA-2 subunit alone or in combination with other KAR subunits in HEK-293 cells. The cell surface expression of the KAR subunit homo- and heteromers were analysed using biotinylation and agonist-stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA-2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA-2 was dramatically increased by co-expression with either of the low-affinity KAR subunits GluR5-7. However, co-expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA-2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA-2 content compared to microsomal ones. Thus, KA-2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.     

Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors

The third intracellular (3i) loops of the alpha 2A- and alpha 2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha 2A, alpha 2B, and alpha 2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha 2-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha 2-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha 2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2) < or = 60 min) apical versus slower (t(1/2) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha 2-AR-interacting region (Sp151-483) by fusion with apically targeted p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 h and eliminated the rapid phase (0-60 min) of alpha 2B-AR turnover on that surface. Furthermore, we examined alpha 2B-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp+/+) or spinophilin knock-out (Sp-/-) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha 2B-AR was accelerated in mouse embryo fibroblasts derived from Sp-/- mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha 2B-AR and presumably all three alpha2-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha 2-ARs to proteins interacting with spinophilin via other domains.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.     

Visualization of cell surface vasopressin V1a receptors in rat hepatocytes with a fluorescent linear antagonist.

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8-PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8-PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8-PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8-PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8-PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8-PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8-PVA. We conclude that Rhm8-PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.     

Induction of V2 receptors in renal medulla of homozygous Brattleboro rats by arginine vasopressin

Homozygous Brattleboro rats display pronounced diabetes insipidus and when treated continuously with arginine vasopressin (AVP) acquire the ability to produce concentrated urine. In this study, the effects of continual AVP replacement on the pharmacological properties of the renal medullary V2 receptor and coupling to adenylate cyclase were examined. Osmotic minipumps that delivered AVP at four different rates were implanted into male homozygous Brattleboro rats. At the end of the 14 day treatment period, urine osmolalities were 280 +/- 24, 474 +/- 105, 1777 +/- 304 and 2202 +/- 175 mOsm/kg H2O for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. Plasma AVP levels were below the level of detection for the 0 and 31.25 ng/hr treatment groups, and were 2.5 +/- 0.5 and 6.5 +/- 1.8 pg/ml for the 62.5 and 125 ng/hr treatment groups. Saturation experiments using [3H] AVP and renal medullary membranes revealed binding site concentrations of 57 +/- 9, 84 +/- 23, 164 +/- 17 and 150 +/- 18 fmol/mg protein for the 0, 31.25, 62.5 and 125 ng/hr treatment groups, respectively. AVP-stimulated cyclic AMP accumulation was enhanced in renal medullary membranes prepared from the 62.5 and 125 ng/hr treatment groups when compared to that in the 0 and 31.25 ng/hr treatment groups. From these results, it appears that circulating AVP is necessary for expression of functional V2 receptors in the homozygous Brattleboro rat renal medulla.     

Mapping the binding site of arginine vasopressin to V1a and V1b vasopressin receptors

Starting from the 2.8-A resolution x-ray structure of bovine rhodopsin, three-dimensional molecular models of the complexes between arginine vasopressin and two receptor subtypes (V1a, V1b) have been built. Amino acid sequence alignment and docking studies suggest that four key residues (1.35, 2.65, 4.61, and 5.35) fine tune the binding of vasopressin and related peptide agonists to both receptor subtypes. To validate these predictions, a series of single or double mutants were engineered at V1a and V1b receptor subtypes and tested for their binding and functional properties. Two negatively charged amino acids at positions 1.35 and 2.65 are key anchoring residues to the Arg8 residue of arginine vasopressin. Moreover, two amino acids (V(4.61) and P(5.35)) delineating a hydrophobic subsite at the human V1b receptor are responsible for the recognition of V1b selective peptide agonists. Last, one of the latter positions (5.35) is hypothesized to explain the pharmacological species differences between rat and human vasopressin receptors for a V1b peptide agonist. Altogether these refined three-dimensional models of V1a and V1b human receptors should enable the identification of further new selective V1a and V1b agonists as pharmacological but also therapeutic tools.     

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase alpha-subunits substituted at the same position

The surface properties of wild-type and six mutant alpha-subunits of tryptophan synthase substituted at the same position, 49, which is buried in the interior, were measured by surface tension, foaming and emulsifying properties to correlate the surface properties with the stabilities. The conformational stabilities of the seven alpha-subunits differed dramatically depending on the characteristics of the substituting residues [Yutani et al. (1987) Proc. Natl. Acad. Sci., 84, 4441-4444]. The mutant proteins substituted by isoleucine and phenylalanine in place of glutamic acid at position 49 were more stable than the other proteins and showed higher surface tension and lower foaming and emulsifying properties than the wild-type and other mutant proteins. Good correlations were observed between these surface properties and values of the Gibbs free energy of unfolding in water, of the proteins. This indicates that the surface properties of the alpha-subunits of tryptophan synthase depend closely on the conformational stabilities.     

Comparison of helix stability in wild-type and mutant LamB signal sequences

Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an alpha-helical conformation in low dielectric environments was a property of functional sequences. We have now used nuclear magnetic resonance to establish further characteristics of the helical conformation of these signal peptides in a solvent mixture (50% trifluoroethanol, by volume, in water) which mimics the conformational distribution of these peptides in lipid vesicles. The interactions of signal sequences in vivo may depend on the location of the helix in the sequence, on the length of the helical segment, and on the stability of the helix. We find that the hydrophobic core has the most persistent helix conformation and that the stability of this helix correlates with in vivo function of different mutants of the LamB signal sequence. In the family of signal peptides studied here, the length of the helix required for function appears to be less rigidly restricted since a signal peptide from a functional pseudorevertant with 4 residues deleted from the hydrophobic core takes up helix as stably as wild type but incorporates fewer residues in the helix.     

Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.     

A cell surface inactive mutant of the human lutropin receptor (hLHR) attenuates signaling of wild-type or constitutively active receptors via heterodimerization

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797–1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.     

Intracellular pathways of V(1) and V(2) receptors activated by arginine vasopressin in rat hippocampal neurons.

To explore the intracellular pathways activated by vasopressin receptors, the effects of arginine vasopressin (AVP) and its analogues mediating glycine (Gly)-induced Cl(-) currents (I(Gly)) were examined in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch recording technique. AVP and its analogues inhibited I(Gly) in a concentration-dependent manner. The inhibitory actions of AVP(4-9) (AVP metabolite) and NC-1900 (AVP(4-9) analogue) were reversed by a V(1) receptor antagonist, or pretreatment with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N', N'-tetraacetic acid. In contrast, these blocking procedures had no effect on the 1-desamino-8-D-AVP (DDAVP; V(2) agonist) action. A V(2) receptor antagonist did not block the inhibitory action of AVP(4-9) or NC-1900, but blocked that of DDAVP. The inhibitory action of AVP was completely blocked by the co-application of the V(1) and V(2) antagonists. The inhibitory action of NC-1900 was not affected by perfusion with a Ca(2+)-free external solution, but was strongly blocked by thapsigargin. The intracellular application of heparin or anti-inositol 1,4,5-triphosphate (IP(3)) also blocked the NC-1900 action. Furthermore, Ca(2+)/calmodulin (CaM) inhibitors blocked the NC-1900 action, while a CaM-dependent kinase II inhibitor and PKC modulators had no effect. 2',5'-Dideoxyadenosine (an adenylate cyclase inhibitor), H-89, and Rp-cAMPS blocked the inhibitory actions of NC-1900 and DDAVP. These results suggest that the activation of the V(1) receptor in the hippocampal neurons induces the production of IP(3), which releases Ca(2+) from the IP(3)-sensitive Ca(2+) storage sites. The Ca(2+) binds to CaM, resulting in the activation of Ca(2+/)CaM-sensitive adenylate cyclases. The activation of protein kinase A through the adenylate cyclase inhibits I(Gly).     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function.

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.