Mutational analysis of the N-glycosylation sites of Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokines (DARC) is a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group antigen. The polypeptide chain of DARC contains two NSS motifs at positions 16 and 27 and one NDS motif at position 33 that represent canonical sequences for efficient N-glycosylation. To verify whether all of these three sites are occupied by a sugar chain, we generated mutants in which potential N-glycosylation sites (AsnXSer) were removed by replacement of serine by alanine. Seven DARC glycosylation variants, missing one (S18A, S29A, S35A), two (S18A.S29A, S18A.S35A, S29A.S35A), or three (S18A.S29A.S35A) glycosylation sites, were obtained. cDNA encoding DARC mutants was cloned into the eukaryotic expression vector pcDNA3.1/myc-HisA and expressed in human K562 cells. Stable transfectants expressing wild-type or mutated forms of Duffy were then lysed, purified by metal-affinity chromatography, and subjected to Western blots with an anti-Duffy monoclonal antibody. The gel electrophoresis data indicate that all three canonical sites are used for sugar attachment.     

Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.     

Synthetic probes for the alpha-factor receptor

The binding of the tridecapeptide yeast mating pheromone, alpha-factor, to its receptor represents an excellent model for the investigation of peptide hormone-receptor interactions. In this paper we present a number of strategies to probe the binding site of the alpha-factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the alpha- or epsilon-amine in [Nle12]-alpha-factor was accomplished using 3-[3,5-diiodo-4-hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton-Hunter reagent). At pH 8.0 in a N-N-dimethylformamide/water mixture the ratio of epsilon- to alpha-acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2-dimethoxyethane/water mixture alpha-acylation was favored by more than 3 to 1. The product distribution was found to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially alpha-acylated [Nle12]-alpha-factor and served as a temporary masking group during the synthesis of epsilon-Bolton-Hunter acylated pheromone. Biotin or diiodo Bolton-Hunter reagents were also directly incorporated into [Nle12]-alpha-factor or Lys[Nle12]-alpha-factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylacetamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton-Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of beta-adrenergic receptor glycosylation

The beta-adrenergic receptor (beta AR) contains significant amounts of N-linked carbohydrate. Deletion mutants spanning the four consensus glycosylation sites on the receptor and single amino acid substitutions within the two amino-terminal consensus glycosylation sites reveal that both the amino-terminal sites are utilized. None of the glycosylation-defective beta AR mutants exhibited altered ligand binding in transient expression assays. In addition, the mutant beta ARs which were completely devoid of carbohydrate were capable of coupling to Gs and stimulating adenylyl cyclase in stable L cell lines. In contrast to the wild-type beta AR, the glycosylation-deficient beta ARs expressed in these cells showed a 50% decrease in the level of accumulation on the cell surface. Therefore, while glycosylation of the beta AR does not appear to be essential for receptor function, it is important for correct trafficking of the beta AR protein through the cell.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii alpha-L-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant alpha-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn(83)-Thr-Thr and Asn(202)-Ser-Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn(83), Asn(202), and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn(202) may contribute to thermostability and catalysis.     

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.     

Mutational analysis of the role of HPr in Listeria monocytogenes

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.     

Mutational analysis of residue roles in AraC function

The previously isolated hemiplegic, induction-negative, repression-positive mutants, H80R and Y82C, were found to be defective in the binding of arabinose. Randomization of other residues close to arabinose in the three-dimensional structure of AraC or that make strong interactions with arabinose yielded induction-negative, repression-positive mutants. The induction and repression properties of mutants obtained by randomizing individual residues of the N-terminal arm of AraC allowed identification of the domain with which that residue very likely makes its predominant interactions. Residues 8-14 of the arm appear to make their predominant interaction with the DNA-binding domain. Although the side-chain of residue 15 interacts directly with arabinose bound to the N-terminal dimerization domain, the properties of mutant F15L indicate that this mutation increases the affinity of the arm for the DNA-binding domain.     

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.     

Mutational analysis of the cellular receptor for poliovirus.

To identify sequences of the cellular poliovirus receptor (PVR) involved in viral infection, mutant PVR cDNAs were constructed and assayed for biological activity in mouse L cells. To confirm that mutant PVRs reached the cell surface, an immunological tag, consisting of part of CH3 from human immunoglobulin G1, was engineered into the PVR. Deletion of PVR amino acids 256 to 320 or 385 to the carboxy terminus yielded receptors that were able to support poliovirus infection. PVRs lacking amino acids 40 to 136 or 137 to 256 were expressed at the cell surface but were not active as receptors for poliovirus. The results show that immunoglobulin-type domain 3 and the extreme carboxy terminus of the PVR are not required for viral receptor function, but sequences within the two amino-terminal domains contribute to the initiation of poliovirus infection.     

Identification of residues that contribute to receptor activation through the analysis of compensatory mutations in the G protein-coupled alpha-factor receptor

The alpha-factor receptor (Ste2p) stimulates mating of the yeast Saccharomyces cerevisiae. Ste2p belongs to the large family of G protein-coupled receptors that are characterized by seven transmembrane alpha-helices. Receptor activation is thought to involve changes in the packing of the transmembrane helix bundle. To identify residues that contribute to Ste2p activation, second-site suppressor mutations were isolated that restored function to defective receptors carrying either an F204S or Y266C substitution which affect residues at the extracellular ends of transmembrane domains 5 and 6, respectively. Thirty-five different suppressor mutations were identified. On their own, these mutations caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function. The majority of the mutations affected residues in the transmembrane segments that are predicted to face the helix bundle. Many of the suppressor mutations caused constitutive receptor activity, suggesting they improved receptor function by partially restoring the balance between the active and inactive states. Analysis of mutations in transmembrane domain 7 implicated residues Ala281 and Thr282 in receptor activation. The A281T and T282A mutants were supersensitive to S. cerevisiae alpha-factor, but were defective in responding to a variant of alpha-factor produced by another species, Saccharomyces kluyveri. The A281T mutant also displayed 8.7-fold enhanced basal signaling. Interestingly, Ala281 and Thr282 are situated in approximately the same position as Lys296 in rhodopsin, which is covalently linked to retinal. These results suggest that transmembrane domain 7 plays a role in receptor activation in a wide range of G protein-coupled receptors from yeast to humans.     

Mutational analysis of the ligand-binding domain of the prolactin receptor

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.     

Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor.

The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family. Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor. The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate. Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist. alpha-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor. The alpha-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants. Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes. These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor. The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.     

Mutational analysis reveals multiple distinct sites within Fc gamma receptor IIB that function in inhibitory signaling

The low-affinity receptor for IgG, FcgammaRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcgammaRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5'- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor's C-terminal 16 residues are also required for detectable FcgammaRIIB association with SHIP in vivo and for FcgammaRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcgammaRIIB-mediated dephosphorylation of CD19 is independent of the cytoplasmic tail distal from residue 237, including the ITIM. Finally, the findings indicate that tyrosines 290, 309, and 326 are all sites of significant FcgammaRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcgammaRIIB contribute uniquely to transduction of FcgammaRIIB-mediated inhibitory signals.     

Down regulation of the alpha-factor pheromone receptor in S. cerevisiae

The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor. Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis. Pheromone-resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation. Our results suggest that alpha-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha-factor. We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor.     

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.     

Mutational analysis of the structure and function of opioid receptors

The cloning of the opioid receptors allows the investigation of receptor domains involved in the peptidic and nonpeptidic ligand interaction and activation of the opioid receptors. Receptor chimera studies and mutational analysis of the primary sequences of the opioid receptors have provided insights into the structural domains required for the ligand recognition and receptor activation. In the current review, we examine the current reports on the possible involvement of extracellular domains and transmembrane domains in the high-affinity binding of peptidic and nonpeptidic ligands to the opioid receptor. The structural requirement for the receptors' selectivity toward different ligands is discussed. The receptor domains involved in the activation and subsequent cellular regulation of the receptors' activities as determined by mutational analysis will also be discussed. Finally, the validity of the conclusions based on single amino acid mutations is examined.     

Role of extracellular charged amino acids in the yeast alpha-factor receptor

The yeast pheromone receptor, Ste2p, is a G protein coupled receptor that initiates cellular responses to alpha-mating pheromone, a 13 residue peptide that carries a net positive charge at physiological pH. We have examined the role of extracellular charged groups on the receptor in response to the pheromone. Substitutions of Asn or Ala for one extracellular residue, Asp275, affected both pheromone binding and signaling, suggesting that this position interacts directly with ligand. The other seven extracellular acidic residues could be individually replaced by polar residues with no detectable effects on receptor function. However, substitution of Ala for each of these seven residues resulted in impairment of signaling without affecting pheromone binding, implying that the polar nature of these residues promotes receptor activation. In contrast, substitution of Ala for each of the six positively charged residues at the extracellular surface of Ste2p did not affect signaling.