Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.     

Intracellularly located misfolded glycoprotein hormone receptors associate with different chaperone proteins than their cognate wild-type receptors

Most loss-of-function mutations of the glycoprotein hormone receptors have been found to be due to the misfolding of the receptor, resulting in its intracellular retention and, therefore, decreased cell surface expression. Chaperone proteins within the endoplasmic reticulum play an essential role in facilitating the folding of newly synthesized proteins and in recognizing and segregating misfolded proteins, thereby preventing their transit to the Golgi. The present study was conducted to begin to elucidate the role of chaperone proteins in the folding of the glycoprotein hormone receptors and misfolded mutants thereof. Toward this end, we examined the potential associations of calnexin, calreticulin, Grp94, BiP, ERp57, and protein disulfide-isomerase with each of the three glycoprotein hormone receptors. Calnexin, calreticulin, and protein disulfide-isomerase were found to associate with the immature forms of all three wild-type (wt) glycoprotein hormone receptors. As examples of misfolded glycoprotein hormone receptors, we studied two human LH receptor (hLHR) loss-of-function mutants that we show to be expressed predominantly as immature forms that are retained intracellularly. Significantly, the patterns of chaperone protein associations with the misfolded hLHR mutants differ from that observed with the wt hLHR. Furthermore, and unexpectedly, the chaperone protein associations were found to differ between the two misfolded hLHR mutants. Altogether, our studies show that although the same chaperone proteins are used by the three wt glycoprotein hormone receptors, different chaperone proteins associate with misfolded mutants thereof, and the specificity of interactions can vary between mutants, most likely reflecting the different stages of folding they achieve before being targeted for degradation.     

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.     

A splice variant of the human luteinizing hormone (LH) receptor modulates the expression of wild-type human LH receptor

We previously reported a splice variant form of human LH receptor [hLHR(exon 9)] that lacks exon 9, coding the N-terminal extracellular region close to the first transmembrane domain. Several recent studies suggest that G protein-coupled receptors are able to form dimerization or oligomerization of the receptor, suggesting an intermolecular interaction between hLHR(exon 9) and the wild-type LH receptor (hLHR). The aim of this study, using coimmunoprecipitation, is to examine whether hLHR forms an association with hLHR(exon 9). An interaction between hLHR(exon 9) with the immature band (68 kDa) of hLHR and not with the mature band (85 kDa) was seen. When hLHR and hLHR(exon 9) were coexpressed, the density of hLHR expression was significantly reduced, compared with hLHR expressed alone. The human chorionic gonadotropin-stimulated cAMP accumulation in the cells expressing hLHR(exon 9) was also impaired, compared with the cells expressing hLHR. In this study, we demonstrated that hLHR is capable of forming receptor complexes. Our findings may expand the possibility of a splice variant of hLHR specifically modulating the functional property of the wild-type hLHR.     

Maturation of Receptor Proteins in Eukaryotic Expression Systems

Abstract

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t_1/2 of 6 h, while the immature form has a t_1/2 of 20 min. In the absence of N-linked glycosylation the t_1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t_1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.     

Truncated activin type II receptor inhibits erythroid differentiation in K562 cells

Two receptor serine/threonine kinases (types I and II) have been identified as signaling transducing activin receptors. We studied the possibility of inhibiting activin A-dependent differentiation in K562 cells, using a dominant negative mutant of type II receptor. A vector was constructed expressing activin type II truncated receptor (ActRIIa) that lacks the cytoplasmic kinase domain. Since activin type I and II receptors form heteromeric complexes for signaling, the mutant receptors compete for binding to endogenous receptors, hence acting in a dominant negative fashion. K562 cells were stably transfected with ActRIIa, and independent clones were expanded. The truncated cDNA was integrated into the genome of the transfectants, as shown by polymerase chain reaction; and the surface expression of truncated receptors was shown by affinity cross-linking with (125)I-activin A. In wild-type K562 cells, activin A induced erythroid differentiation and cells started to express hemoglobins. In transfected cells expressing ActRIIa, the induction of erythroid differentiation was abrogated and less than 10% of cells were hemoglobin-containing cells after culture with activin A. Further transfection with wild-type type II receptors rescued the mutant phenotype of these transfectants, indicating that the effect of ActRIIa is dominant negative. In addition, phosphorylation of the cytoplasmic kinase domain of the type II receptor in vitro confirms the autophosphorylation of this portion of the receptor. Therefore, induction of erythroid differentiation in vitro is mediated through the cell surface activin receptor, and interference with this receptor signaling inhibits this process of differentiation in K562 cells.     

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.     

The association of arrestin-3 with the human lutropin/choriogonadotropin receptor depends mostly on receptor activation rather than on receptor phosphorylation.

Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the beta2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion.     

Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.     

Retention of glucose on oligosaccharide chains linked to the LH/hCG receptor prevents cell surface expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.     

Chemical cross-linking of heteromeric glucocorticoid receptors

Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.     

Differential regulation of the human adrenocorticotropin receptor [melanocortin-2 receptor (MC2R)] by human MC2R accessory protein isoforms alpha and beta in isogenic human embryonic kidney 293 cells

The ACTH receptor [melanocortin-2 receptor (MC2R)] is the smallest known G protein-coupled receptor (GPCR). Herein, human MC2R accessory protein (MRAP) isoforms alpha and beta, cloned from a human fetal adrenal gland, were expressed with c-Myc-tagged MC2R (Myc-MC2R) in 293/Flp recombinase target site cells by homologous recombination. Although insertion of Myc-MC2R at the plasma membrane occurred without MRAP assistance, ACTH stimulation of cAMP production was only detected in cells coexpressing MC2R with either MRAP isoform. On the other hand, a MC2R-green fluorescent protein fusion transfected with either MRAPalpha or MRAPbeta was impaired both in cell membrane localization and signaling. MRAP isoforms were also tagged with either Flag or 6xHis epitopes. In cell populations coexpressing transiently and/or stably Myc-MC2R with MRAPalpha or MRAPbeta, stimulation with ACTH induced production of cAMP with EC(50) values lower in MRAPalpha- than in MRAPbeta-expressing cells. ACTH only bound Myc-MC2R in the presence of MRAP. Higher Myc-MC2R cell surface density was observed in the presence of MRAPbeta comparatively to MRAPalpha, possibly contributing to higher ACTH binding capacity and higher maximal cAMP responses observed in MRAPbeta-expressing cells. Immunofluorescence studies indicated that MRAP isoforms were localized near the plasma membrane and in the vicinity, but not colocalized, with Myc-MC2R. In summary, through the generation of a new all-human experimental model devoid of endogenous MCRs, we present evidence that human MRAP isoforms, although not essential for MC2R localization at the plasma membrane, are essential for ACTH binding and ACTH-induced cAMP production and that they differentially regulate, although modestly, cell membrane density and functional properties of MC2R.     

Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.     

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.     

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.     

Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C

Mutations in the gene encoding SP-C (surfactant protein C; SFTPC) have been linked to interstitial lung disease (ILD) in children and adults. Expression of the index mutation, SP-C(Deltaexon4), in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of endoplasmic reticulum (ER) stress pathways, and cytotoxicity. In this study, we show that stably transfected cells adapted to chronic ER stress imposed by the constitutive expression of SP-C(Deltaexon4) via an NF-kappaB-dependent pathway. However, the infection of cells expressing SP-C(Deltaexon4) with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response, and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to environmental insults that ultimately overwhelm the homeostatic cytoprotective response.     

Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing

Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface.     

Involvement of bovine leukemia virus in induction and inhibition of apoptosis

In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G, that has an enhanced capacity to transactivate the long terminal repeat (LTR) of BLV and the cellular proto-oncogene c-fos when compared with wild-type Tax (wt-Tax). We demonstrate here that an infectious strain of BLV containing the mutant D247G form of Tax also differs in its capacity to modulate cell survival both positively and negatively. When peripheral blood mononuclear cells (PBMCs) infected with wild-type or mutant BLV are cultured ex vivo with staurosporine, an agent known to induce a mitochondrial caspase cascade pathway regulating apoptosis, the rate of apoptosis is reduced to a greater extent in cells infected with mutant BLV than wild-type BLV, consistent with previous observations in cultures without staurosporine. The increase in survival was associated with an increase in expression of mRNA of bcl-xl but not bcl-2 and bax ex vivo. In contrast, when a tissue culture-adapted cell line, 293T, was transiently transfected with either wild-type or mutant BLV, apoptosis was induced. The increase in the rate of apoptosis was higher in cells transfected with mutant BLV. The same difference was noted in cells transiently transfected with wild-type and mutant D247G Tax, suggesting that the observed positive and negative modulation of cell survival is attributed to the functional characteristics of mutant D247G Tax.