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PubMed1987

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein.

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.

Amino Acid Sequence↗
PubMed1996

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity.

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.

Amino Acid Sequence↗
PubMed2000

The role of tryptophan residues in the 5-Hydroxytryptamine(3) receptor ligand binding domain.

Aromatic amino acids are important components of the ligand binding site in the Cys loop family of ligand-gated ion channels. To examine the role of tryptophan residues in the ligand binding domain of the 5-hydroxytryptamine(3) (5-HT(3)) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT(3A) receptor subunit to tyrosine or serine. The mutants were expressed as homomeric 5-HT(3A) receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Trp(90), Trp(183), and Trp(195) to tyrosine resulted in functional receptors, although with increased EC(50) values (2-92-fold) to 5-HT(3) receptor agonists. Changing these residues to serine either ablated function (Trp(90) and Trp(183)) or resulted in a further increase in EC(50) (Trp(195)). Mutation of residue Trp(60) had no effect on ligand binding or receptor function, whereas mutation of Trp(95), Trp(102), Trp(121), and Trp(214) ablated ligand binding and receptor function, and all but one of the receptors containing these mutations were not expressed at the plasma membrane. We propose that Trp(90), Trp(183), and Trp(195) are intimately involved in ligand binding, whereas Trp(95), Trp(102), Trp(121), and Trp(214) have a critical role in receptor structure or assembly.

Amino Acid Sequence↗
PubMed1998

Angiotensin AT1 receptor subtypes in the rabbit pulmonary artery. A ligand binding study.

The present ligand binding study showed that the rabbit pulmonary artery contained two subtypes of losartan-sensitive angiotensin receptor. The two receptor subtypes are differentially distributed. The high affinity receptor subtype is located predominantly in the cardia end of the artery while the low affinity receptor subtype is found mainly in the pulmonary portion of the artery. The Kd for the high and low affinity receptors for sar1, Ile8-angiotensin II was found to be 0.25 +/- 0.005 and 0.88 +/- 0.02 nM, respectively; and for angiotensin II to be 0.43 +/- 0.001 and 0.96 +/- 0.02 nM, respectively. In the presence of 1 mM GppNHp, the high affinity receptor subtype was converted to the low affinity subtype, indicating that it is G-protein coupled. 1 mM GppNHp had no effect on the low affinity receptor subtype. The present data support the findings of an earlier functional study which also showed the existence and similar differential distribution of two losartan-sensitive angiotensin receptors in the rabbit pulmonary artery. However, the significance of these findings in regard to the regulation of pulmonary circulation in normal and pathological conditions remains to be investigated.

Angiotensin I↗
PubMed2005

Kinetics of allosteric conformational transition of a macromolecule prior to ligand binding: analysis of stopped-flow kinetic experiments.

Two fundamentally different mechanisms of ligand binding are commonly encountered in biological kinetics. One mechanism is a sequential multistep reaction in which the bimolecular binding step is followed by first-order steps. The other mechanism includes the conformational transition of the macromolecule, before the ligand binding, followed by the ligand binding process to one of the conformational states. In stopped-flow kinetic studies, the reaction mechanism is established by examining the behavior of relaxation times and amplitudes as a function of the reactant concentrations. A major diagnostic tool for detecting the presence of a conformational equilibrium of the macromolecule, before the ligand binding, is the decreasing value of one of the reciprocal relaxation times with the increasing [ligand]. The sequential mechanism cannot generate this behavior for any of the relaxation times. Such dependence is intuitively understood on the basis of approximate expressions for the relaxation times that can be comprehensively derived, using the characteristic equation of the coefficient matrix and polynomial theory. Generally, however, the used approximations may not be fulfilled. On the other hand, the two kinetic mechanisms can always be distinguished, using the approach based on the combined application of pseudo-first-order conditions, with respect to the ligand and the macromolecule. The two experimental conditions differ profoundly in the extent of the effect of the ligand on the protein conformational equilibrium. In a large excess of the ligand, the conformational equilibrium of the macromolecule, before the ligand binding, is strongly affected by the binding process. However, in a large excess of the macromolecule, ligand binding does not perturb the internal equilibrium of the macromolecule. As a result, the normal mode, affected by the conformational transition, is absent in the observed relaxation process. In the case of a sequential mechanism, the number of relaxation times is not altered by different pseudo-first-order conditions. Thus, the approach provides a strong diagnostic criterion for detecting the presence of the conformational transition of the macromolecule and establishing the correct mechanism. Application of this approach is illustrated for the binding of 3'-O-(N-methylantraniloyl)-5'-diphosphate to the E. coli DnaC protein.

Adenosine Diphosphate↗
PubMed1997

Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

Animals↗
PubMed2000

Determination of pK(a) values of carboxyl groups in the N-terminal domain of rat CD2: anomalous pK(a) of a glutamate on the ligand-binding surface.

The ligand-binding surface of the T-lymphocyte glycoprotein CD2 has an unusually high proportion of charged residues, and ionic interactions are thought to play a significant role in defining the ligand specificity and binding affinity of CD2 with the structurally homologous ligands CD48 (in rodents) and CD58 (in humans). The determination of the electrostatic properties of these proteins can therefore contribute to our understanding of structure-activity relationships for these adhesion complexes that underpin T-cell adhesion to antigen-presenting cells. In this study, we investigated the pH titration behavior of the carboxyl groups of the N-terminal domain of rat CD2 (CD2d1) using the chemical shifts of backbone amide nitrogen-15 ((15)N) and proton NMR resonances, and carboxyl carbon-13 ((13)C) signals. The analysis revealed the presence of a glutamate (Glu41) on the binding surface of rat CD2 with an unusually elevated acidity constant (pK(a) = 6.73) for CD2d1 samples at 1.2 mM concentration. pH titration of CD2d1 at low protein concentration (0.1 mM) resulted in a slight decrease of the measured pK(a) of Glu41 to 6.36. The ionization of Glu41 exhibited reciprocal interactions with a second glutamate (Glu29) in a neighboring location, with both residues demonstrating characteristic biphasic titration behavior of the carboxyl (13)C resonances. Measurements at pH 5.5 of the two-bond deuterium isotope shift for the (13)C carboxyl resonances for Glu41 and Glu29 [(2)DeltaC(delta)(O(epsilon)D) = 0.2 and 0.1 ppm, respectively] were consistent with the assignment of the anomalous pK(a) to Glu41, under the strong influence of Glu29. The characterization of single site mutations of CD2d1 residues Glu41 and Glu29 to glutamine confirmed the anomalous pK(a) for Glu41, and indicated that electrostatic interaction with the Glu29 side chain is a significant contributing influence for this behavior in the wild-type protein. The implications of these observations are discussed with respect to recent structural and functional analyses of the interaction of rat CD2 with CD48. In particular, CD2 Glu41 must be a candidate residue to explain the previously reported strong pH dependence of binding of these two proteins in vitro.

Animals↗
PubMed2001

Dissection of receptor folding and ligand-binding property with functional minireceptors of LDL receptor-related protein.

The LDL receptor-related protein (LRP) is a large, multifunctional endocytic receptor that binds and endocytoses a variety of structurally and functionally distinct ligands. LRP contains four putative ligand-binding domains. However, only domains II, III and IV, but not domain I, bind the receptor-associated protein (RAP), a molecular chaperone and universal antagonist for LRP. In order to dissect the function of RAP in LRP folding and to examine the ligand-binding properties of LRP, we generated LRP minireceptors that represent each of the four putative ligand-binding domains (termed mLRP1, mLRP2, mLRP3 and mLRP4, respectively). We found that proper folding and trafficking of mLRP2, mLRP3, mLRP4, but not mLRP1, is facilitated by coexpression of RAP. When these mLRPs were stably expressed in Chinese Hamster Ovary cells that lack the endogenous LRP, we found that each of these receptors was processed and traffics through the secretory pathway. Cell surface expression of these minireceptors was quantitatively examined by flow cytometric analyses. Using these minireceptor cell lines to map the ligand-binding domains, we found that although the majority of LRP ligands bind to both domain II and domain IV, Pseudomonas exotoxin A utilizes only domain IV for its binding to LRP. We conclude that while domains II and IV of LRP share many ligand-binding properties, each of the putative ligand-binding domains of LRP is unique in its contribution to ligand binding.

Animals↗
PubMed2001

Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding.

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.

Amino Acid Motifs↗
PubMed2000

The important role of residue F268 in ligand binding by LXRbeta.

Liver X receptors (LXRs) are nuclear receptors that regulate the metabolism of cholesterol and bile acids. Despite information on the specificity of their natural ligands, oxysterols, relatively little is known about the ligand binding site in LXRs. The helix 3 region in the ligand binding domain (LBD) of peroxisome proliferator-activated receptors (PPARs) has been implicated in ligand entry. Sequence alignment of LXRs, farnesoid X receptor (FXR), and PPARs identified the corresponding helix 3 region in the LXRbeta LBD. Residues F268 and T272, which are conserved in all the aligned sequences and only in LXRs and FXR, respectively, were replaced with alanine. The effects of these mutations on ligand binding and receptor activation were examined using an in vitro ligand binding assay and a cell based reporter assay, respectively. The LXRbeta mutant F268A did not bind ligand. In contrast, conversion of T272 to alanine has no effect on ligand binding. By transiently expressing a chimeric receptor containing Escherichia coli tetracycline repressor (TetR) and LXRbeta LBD and a reporter with a TetR binding site, we show that mutant F268A lost the ability to activate transcription of the reporter, whereas mutant T272A still has an activity similar to that of the wild-type LXRbeta. These data, consistent with the findings in the in vitro ligand binding assay and our 3D modeling, are the first study that identifies a residue critical for ligand binding in LXRbeta.

Amino Acid Sequence↗
PubMed1992

Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures.

We have isolated a set of ligand-binding DNA sequences from a large pool of random sequence DNAs by selection and amplification in vitro, using similar methods to those described for the isolation of ligand-binding RNAs. The ligand-DNA interactions are both sequence- and ligand-specific, and are dependent on proper folding of the single-stranded DNA. Some ligands led to the isolation of more DNA sequences than RNA sequences, and vice versa. Analysis of individual sequences reveals that ligand binding is DNA-specific; RNAs of identical sequence could not interact with the same ligands. Ligand-binding DNAs might be more suitable than RNAs as potential pharmacological reagents because of the greater stability of DNA. The apparent primacy of RNA in the early evolution of life may have been due to its availability rather than to its functional superiority.

Base Composition↗
PubMed1997

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Correlation with the structure of ligands and the structure of binding protein.

Ligands that are transported by the maltose transport system of Escherichia coli must first bind to the periplasmic maltose-binding protein (MBP). However, binding of a ligand does not always lead to its transport. As reported earlier, reduced or oxidized maltodextrins bind tightly to MBP but are not transported; some mutant MBPs, such as MalE254, bind maltodextrins tightly but cannot produce their transport. In this study, UV differential spectroscopy and fluorescence emission spectroscopy were used to study the modes by which various ligands bind to MBP. Maltose binding produced a red shift in the fluorescence emission spectrum of wild type MBP and a sharp hypochromatic trend below 265 nm in its UV spectrum (R mode (for red)). On the other hand, binding of reduced, oxidized, or cyclic maltodextrins produced a pronounced blue shift in the fluorescence emission spectrum of wild type MBP and a peak at about 250 nm in its UV difference spectrum (B mode (for blue). Binding of reducing maltodextrins to wild type MBP produced spectral changes that seemed to be a mixture of predominantly R mode binding and some B mode binding, whereas their binding to mutant MBP MalE254 produced changes indicative of pure B mode binding. Thus, the ligands that are bound exclusively via the B mode to either the wild type or MalE254 MBP are not transported.

ATP-Binding Cassette Transporters↗
PubMed2005

Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry.

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.

Animals↗
PubMed2006

The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding.

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

Amino Acid Sequence↗
PubMed1998

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1.

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate >> ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.

Animals↗
PubMed1996

Modulation of ligand binding to components of the GABAA receptor complex by ammonia: implications for the pathogenesis of hyperammonemic syndromes.

The effects of 5-2500 microM concentrations of neutral ammonium salts on the binding of ligands to components of the GABAA receptor complex were investigated. [3H]Flunitrazepam binding to the benzodiazepine receptor was enhanced by ammonium (10-500 microM), but not sodium tartrate with EC50 = 98 microM and Emax = 31%. Further increasing ammonium tartrate concentrations (500-2500 microM) decreased [3H]flunitrazepam binding to control levels. The ammonium tartrate-induced increase in [3H]flunitrazepam binding was manifested as a 50% decrease in Kd. Furthermore, GABA increased the potency of ammonium tartrate in enhancing [3H]flunitrazepam binding by 63%. [3H]Ro 15-1788 and [3H]Ro 15-4513 binding to the benzodiazepine receptor was not significantly enhanced by ammonium tartrate (Emax approximately 13%). Ammonium tartrate also increased, then decreased the binding of 500 nM [3H]muscimol to the GABAA receptor (EC50 = 52 microM, Emax = 30%) in a concentration-dependent manner, but had no effect on [3H]SR 95-531 binding (Emax < 16%). The ammonium tartrate-induced alterations in [3H]muscimol binding were demonstrated in saturation assays as the loss of the high affinity binding site and a 27% increase in the Bmax of the low affinity binding site. These results indicate that ammonia biphasically enhances, then returns ligand binding to both the GABA and benzodiazepine receptor components of the GABAA receptor complex to control levels in a barbiturate-like fashion. This suggests that ammonia may enhance GABAergic neurotransmission at concentrations commonly encountered in hepatic failure, an event preceding the suppression of inhibitory neuronal function observed at higher (> 1 mM) ammonia concentrations. This increase in GABAergic neurotransmission is consistent with the clinical picture of lethargy, ataxia and cognitive deficits associated with liver failure and congenital hyperammonemia.

Ammonia↗
PubMedDate not supplied

[Kinetics of ligand binding to nucleic acids at random fillings].

Ligand binding with nucleic acids is described in frames of the theory of random processes. It is shown that the probabilistic description of binding of a ligand to nucleic acid allows one to describe not only the kinetics of changes in the number of bound ligands at arbitrary fillings but also to calculate stationary values of the number of bound ligands and its dispersion. A general analysis of absorption isotherms and the kinetics of ligand binding with nucleic acids allows one to determine the rate constants of formation and decomposition of the ligand-nucleic acid complex. A comparison of the results obtained with the case of low fillings is conducted.

Kinetics↗
PubMed1983

Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor.

The ligand-binding activity of the galactose/N-acetylgalactosamine-specific receptor (Gal/GalNAc receptor) present on the surface of hepatocytes can be modulated under a number of conditions in the intact cell. The carboxylic acid ionophores monensin and nigericin inhibit endocytosis by the Gal/GalNAc receptor in a concentration-dependent manner. Monensin at a concentration of 100 microM reduces the number of binding sites for asialo-orosomucoid and a tri-branched glycopeptide (F2) 5-10-fold; however, the number of Gal/GalNAc receptor subunits detected at the cell surface by a competitive radioimmunoassay and by immunoprecipitation of surface labeled receptor is not significantly altered. Replacement of NaCl in the medium with either N-methylglucamine or sorbitol to isotonicity also inhibits binding and endocytosis. The monensin, nigericin, N-methylglucamine, and sorbitol treatments have in common the ability to alkalinize the cytosol of the hepatocyte. None of these agents has any effect on binding by the isolated Gal/GalNAc receptor nor is the intracellular pH shift of such a magnitude that it would alter binding by the isolated Gal/GalNAc receptor. This has led us to conclude that the ligand-binding properties of the Gal/GalNAc receptor at the cell surface can be modulated in a transmembrane fashion by events other than those involving pH or Ca2+ regulation at the ligand-binding site itself. Such transmembrane modulation of ligand binding by the Gal/GalNAc receptor may provide a rapid and efficient mechanism for mediating ligand release and immediate return of the receptor to the cell surface.

Acetylgalactosamine↗