High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [125I-TyrO]oCRF or [3H-Leu9]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic alpha-helical character of rUcn when compared to oCRF and the chimeric peptides.     

Novel high-affinity photoactivatable antagonists of corticotropin-releasing factor (CRF) photoaffinity labeling studies on CRF receptor, type 1 (CRFR1).

Novel photoactivatable antagonists of human/rat corticotropin-releasing factor (h/rCRF) have been synthesized and characterized. The N-terminal amino acid D-phenylalanine in astressin ?cyclo(30-33) [D-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12-41)?, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I-labeled Tyr0 ovine CRF ([125I-labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF-stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y-79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB-cyclo(30-33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF-(13-41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB-cyclo(30-33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF-(13-41) (compound 2) and exhibited higher inhibition of oCRF-stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells) or Y-79 cells. A highly glycosylated, 66-kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I-labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).     

Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.     

Pharmacological characterization of recombinant rat corticotropin releasing factor binding protein using different sauvagine analogs

Little is known on the structural ligand requirements for corticotropin-releasing factor binding protein (CRFBP) of the rat used as an important experimental animal. To obtain such information recombinant rat CRFBP was produced in stably transfected HEK 293 cells. The primary structure and posttranslational processing of purified rat CRFBP was established by peptide mapping using HPLC combined with mass spectrometric analysis. Rat CRFBP was pharmacologically characterized employing a competition binding assay with tritium-labeled rat urocortin. The rank order of declining affinity of various CRF analogs was urotensin-I, human/rat CRF (h/rCRF), rat urocortin, sauvagine (Svg), and ovine CRF in agreement with the rank order found for human CRFBP. In contrast to astressin, the CRF receptor 2-selective antagonist anti-sauvagine-30 did not show any detectable specific binding to rat CRFBP. The significance of residues 10 to 12 and 21 to 24 of Svg for its low affinity binding was established by changing these residues of Svg to those of h/rCRF. The corresponding residues 22 to 25 of h/rCRF represented the ARAE motif determined to be crucial for binding in agreement with reported data on human CRFBP. Residues 11 to 13 of CRF introduced into Svg also enhanced the affinity to rat CRFBP.     

Rapid production of recombinant human IgG With improved ADCC effector function in a transient expression system

Rapid production of recombinant human IgG with improved antibody dependent cell‐mediated cytotoxicity (ADCC) effector function is presented. The technique employs transient expression of IgG in suspension growing HEK‐293F cells in the presence of the glycosidase inhibitor kifunensine. The procedure takes ～7 days, provided that expression plasmids encoding the IgG of interest are available. Kifunensine inhibits the N‐linked glycosylation pathway of HEK‐293F cells in the endoplasmatic reticulum, resulting in IgG with oligomannose type glycans lacking core‐fucose. IgG1 transiently produced in kifunensine‐ treated HEK‐293F cells has improved affinity for the FcγRIIIA molecule as measured in an ELISA based assay, and almost eightfold enhanced ADCC using primary peripheral blood mononuclear effector cells. Biotechnol. Bioeng. 2010; 105: 350–357. © 2009 Wiley Periodicals, Inc.     

The ligand-selective Domains of Corticotropin-Releasing Factor Type 1 and Type 2 Receptor Reside in Different Extracellular Domains

The nonselective human corticotropin-releasing factor (hCRF) receptor 1 (hCRFR1) and the ligand-selective Xenopus CRFR1 (xCRFR1), xCRFR2, and hCRFR2alpha were compared. To understand the interactions of hCRF, ovine CRF (oCRF), rat urocortin (rUcn), and sauvagine, ligands with different affinities for type 1 and type 2 CRFRs, chimeric and mutant receptors of hCRFR1, xCRFR1, hCRFR2alpha, and xCRFR2 were constructed. In cyclic AMP stimulation and CRF-binding assays, it was established that different extracellular regions of CRFR1 and CRFR2 conferred their ligand selectivities. The ligand selectivity of xCRFR1 resided in five N-terminal amino acids, whereas the N-terminus of both CRFR2 proteins did not contribute to their ligand selectivities. Chimeric receptors in which the first extracellular domain of hCRFR1 replaced that of hCRFR2alpha or xCRFR2 showed a similar pharmacological profile to the two parental CRFR2 molecules. Chimeric receptors carrying the N-terminal domain of xCRFR1 linked to hCRFR2alpha or xCRFR2 displayed a novel pharmacological profile. hCRF, rUcn, and sauvagine were bound with high affinity, whereas oCRF was bound with low affinity. Furthermore, when three or five residues of xCRFR1 (Gln76, Gly81, Val83, His88, Leu89; or Gln76, Gly81, Val83) were introduced into receptor chimeras carrying the N-terminus of hCRFR1 linked to xCRFR2, the same novel pharmacology was observed. These data indicate a compensation mechanism of two differentially selecting regions located in different domains of both xCRFR1 and CRFR2.     

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.     

Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC(50) values as measured by stimulation of [(35)S]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11)in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i) -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.     

Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.     

Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor

Abstract: Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with ∼81% identity to the α-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the β-variant of CRF-R2 existed in Xenopus laevis . Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22-fold) higher affinities than ovine CRF ( K _D = 31.7 n M ) and sauvagine ( K _D = 51.4 n M ). In agreement with these binding data, EC₅₀ values of 39.7 and 1.1 n M were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.     

Serine 331 is the major site of receptor phosphorylation induced by agents that activate protein kinase G in HEK 293 cells overexpressing thromboxane receptor alpha

Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.     

Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312

It has been shown previously that chicken ovalbumin synthesized and secreted in a heterologous cell system is glycosylated at the correct site and that the oligosaccharides at that site, similar to the protein made in hen oviduct, are predominantly of the hybrid type (Sheares, B. T., and Robbins, P. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1993-1997). This site-specific glycosylation of Asn293, but not Asn312, suggested a prominent role for the nascent protein chain rather than the specific cell type in directing the proper attachment of oligosaccharide chains. In the present study, the effect of glycosylation at Asn293 on the glycosylation of Asn312 has been investigated. Using a 20-base oligodeoxynucleotide primer containing a 2-base mismatch, the codon for Asn293 in the chicken ovalbumin gene (AAC) was changed to that for Gln (CAA), thereby preventing glycosylation at amino acid 293. Constructions containing this mutation were transfected into mouse L (tk-) cells which were subsequently labeled with [35S]methionine. Ovalbumin secreted by these cells was recovered by immunoaffinity chromatography and analyzed for the presence of an oligosaccharide attached at Asn312. Treatment of the material with peptide:N-glycosidase F demonstrated that ovalbumin molecules containing Gln substituted for Asn293 were not glycosylated. This further supports our earlier hypothesis that the nascent protein chain is responsible for directing site-specific glycosylation of ovalbumin, and that the presence of an oligosaccharide chain at the first site has no influence on glycosylation at the second site.     

Direct identification of the agonist binding site in the human brain cholecystokininB receptor.

In investigating the agonist binding site of the human brain cholecystokininB receptor (CCKBR), we employed the direct protein chemical approach using a photoreactive tritiated analogue of sulfated cholecystokinin octapeptide, which contains the p-benzoylbenzoyl moiety at the N-terminus, followed by purification of the affinity-labeled receptor to homogeneity. This probe bound specifically, saturably, and with high affinity (KD = 1.2 nM) to the CCKBR and has full agonistic activity. As the starting material for receptor purification, we used stably transfected HEK 293 cells overexpressing functional CCKBR. Covalent labeling of the WGA-lectin-enriched receptor revealed a 70-80 kDa glycoprotein with a protein core of about 50 kDa. Identification of the agonist binding site was achieved by the application of subsequent chemical and enzymatical cleavage to the purified receptor. A radiolabeled peptide was identified by Edman degradation amino acid sequence analysis combined with MALDI-TOF mass spectrometry. The position of the radioactive probe within the identified peptide was determined using combined tandem electrospray mass spectrometry and peptide mapping. The probe was covalently attached within the sequence L52ELAIRITLY61 that represents the transition between the N-terminal domain and predicted transmembrane domain 1. Using this interaction as a constraint to orientate the ligand within the putative receptor binding site, a model of the CCK-8s-occupied CCKBR was constructed. The hormone was found to be placed in a binding pocket built from both extracellular and transmembrane domains of CCKBR with its N-terminus mainly interacting with residues Arg57 and Tyr61.     

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

Sema4C及其相互作用蛋白GIPC的亚细胞定位及荧光共定位研究

目的：观察脑信号蛋白Sema4C及其相互作用蛋白GIPC的亚细胞定位及两者的荧光共定位情况,为明确Sema4C和GIPC在亚细胞水平的相互作用提供佐证。方法：将Sema4C的基因编码区全长、胞外段和胞内段分别构建到pEGFPNl和pEGFPCI表达载体中,将GIPC编码区基因构建到pDsRed-C1表达载体中,分别转染HEK293细胞,观察亚细胞定位;将pEGFPNl-Sema4C和pDsRed-GIPC分别共转染HEl（293和COS7细胞,观察两者的荧光共定位情况。结果：酶切鉴定及测序结果表明重组载体构建正确,Sema4C蛋白全长和胞外段呈跨膜分布,而胞内段在全细胞中呈弥散样分布;GIPC在胞浆内呈斑块状聚集分布;pEGFPNl-Sema4C和pDsRed-GIPC存在荧光共定位区域。结论：Sema4C主要在胞膜和胞浆内表达,GIPC主要在胞浆内呈斑块样聚集分布;Sema4C和GIPC之间存在荧光共定位。     

Sorting nexin 17, a non-self-assembling and a PtdIns(3)P high class affinity protein, interacts with the cerebral cavernous malformation related protein KRIT1

The mammalian sorting nexin (SNX) proteins are involved in the endocytosis and the sorting machinery of transmembrane proteins. Additionally to the family defining phox homology (PX) domain, SNX17 is the only member with a truncated FERM (4.1, ezrin, radixin, and moesin) domain and a unique C-terminal region (together designated as FC unit). By gel filtration and lipid overlay assays we show that SNX17 is a non-self-assembling and a PtdIns(3)P high class affinity protein. A SNX17 affinity to any other phosphoinositides was not detected. By yeast two-hybrid- and GST-trapping assays we identified KRIT1 (krev1 interaction trapped 1) as a new specific interaction partner of the FC unit of SNX17. KRIT1 binds SNX17 by its N-terminal region like the known interaction partner ICAP1alpha (integrin cytoplasmic domain-associated protein-1). The interaction was also detected in HEK 293 cells transiently expressing GFP-tagged KRIT1 and Xpress-tagged SNX17. KRIT1 mutations cause cerebral cavernous malformation (CCM1). Our finding suggests a SNX17 involvement in the indicated KRIT1 function in cell adhesion processes by integrin signaling.     

Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification

Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors. The kinase domain of CHK (amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated. Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation. The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active. A simple plate assay to identify colonies of P. pastoris expressing the recombinant protein is also presented.