Rat Hepatic Lectin Research Articles

Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

The lobular expression of the rat asialoglycoprotein receptor is regulated at the posttranscriptional level

The purpose of this study was to define the distribution of the asialoglycoprotein receptor (ASGP-R) main peptide, rat hepatic lectin (RHL)-1, within the rat liver lobule and to investigate its possible modulation in physiological states characterised by marked changes of receptorial expression. In particular, we chose livers from rats partially hepatectomised or at the end of pregnancy, as models, respectively, of decreased or increased expression of the ASGP-R, and used the in situ hybridisation and immunocytochemistry techniques to analyse in parallel the lobular distributions of RHL-1 mRNA and protein. In normal rat liver, although the RHL-1 mRNA was homogeneously distributed, the RHL-1 peptide was predominantly localised on the surface of pericentral hepatocytes with a gradient of expression towards the periportal zone. This gradient of expression of RHL-1 peptide was reduced in regenerating livers, in which the positive stain was restricted to a few layers of cells around the central vein. In contrast, livers at the end of pregnancy showed an overall increase of the peptide with a concomitant flattening of the gradient across the liver plate. In all the conditions, we never observed important changes in the pattern of expression of the specific mRNA. These findings indicate that the distribution of ASGP-R is heterogeneous across the liver lobule, with a pattern of expression prevalently modulated at the posttranscriptional level.

Closely Related Mammals Have Distinct Asialoglycoprotein Receptor Carbohydrate Specificities

We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.

The RHL-1 subunit of the asialoglycoprotein receptor of thyroid cells: cellular localization and its role in thyroglobulin endocytosis

The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.

A monoclonal antibody to rat asialoglycoprotein receptor that recognizes an epitope specific to its major subunit

We previously developed three mouse monoclonal antibodies (MoAbs) identifying the domain-specific plasma membrane proteins of rat hepatocytes. Based on the results of immunohistochemical and immunoblot analysis, one of them, clone 8D7, likely recognizes the rat asialoglycoprotein receptor (ASGPR), which consists of three polypeptide subunits: rat hepatic lectin (RHL)-1, RHL-2 and RHL-3. In this study, we examined the reactivity of MoAb 8D7 with these subunits in order to identify specifically the protein it recognizes. RHL-1 and RHL-2/3 were expressed in COS-1 mammalian kidney cells transfected with cloned cDNAs. Reactivity of MoAb 8D7 with these proteins was examined by flow cytometry, immunoblotting, and an immunofluorescence staining. By flow cytometry, MoAb 8D7 reacted with cell surface proteins on the COS-1 cells transfected with RHL-1 cDNA but not with RHL-2/3 cDNA. By immunoblotting, the antibody recognized proteins (molecular weights 45 and 96 kDa) in RHL-1-expressing cells but not in the RHL-2/3 cDNA-transfected cells. Immunoreactive staining with MoAb 8D7 was observed on the cell surface and in the cytoplasm of RHL-1-transfected COS-1 cells but not in cells expressing RHL-2/3. These findings indicate that MoAb 8D7 recognizes a subunit-specific epitope on RHL-1 of rat ASGPR.

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor’s lectin properties

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca(2+) and receptor acylation. Binding of (125)I-lactoferrin and (125)I-ASOR to isolated rat hepatocytes at 4 degrees C decreased sharply at pH<6, following similar titration curves. Binding of (125)I-lactoferrin and (125)I-ASOR to hepatocytes was Ca(2+)-dependent. Binding increased progressively at > or =300 microM CaCl(2), in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of (125)I-lactoferrin and (125)I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of (125)I-ASOR binding as well as the complete loss of specific (125)I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca(2+), and also requires the receptor subunits to be acylated.

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca2+ and receptor acylation. Binding of 125I-lactoferrin and 125I-ASOR to isolated rat hepatocytes at 4 °C decreased sharply at pH &amp;lt; 6, following similar titration curves. Binding of 125I-lactoferrin and 125I-ASOR to hepatocytes was Ca2+-dependent. Binding increased progressively at ≥ 300 μM CaCl2, in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of 125I-lactoferrin and 125I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of 125I-ASOR binding as well as the complete loss of specific 125I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca2+, and also requires the receptor subunits to be acylated.

The rat hepatic lectin-1 subunit of the asialoglycoprotein receptor is upregulated by thyrotropin and downregulated by neoplastic transformation of thyroid cells.

We have recently shown that the rat hepatic lectin (RHL)-1 subunit of the asialoglycoprotein receptor (ASGPr) is expressed in the PC C13 differentiated thyroid cell line. To investigate in vivo the expression of RHL-1 and the ability of thyrotropin (TSH) to modulate its expression, reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot assays have been performed on thyroid extracts from rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels. It is shown that RHL-1 expression is down-regulated by T4 (which decreases serum TSH) and upregulated by PTU (which increases serum TSH), at both mRNA and protein levels. The sensitivity of RHL-1 to neoplastic transformation of thyroid cells has been investigated. The RHL-1 expression pattern has been studied in PC C13 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Western blot assays show that RHL-1 expression progressively decreases as PC C13 cells acquire a more transformed phenotype. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a housekeeping gene used as internal control to normalize RHL-1 mRNA content, exhibits no variations in the different PC C13 cell lines used. In addition, we show that both native and asialo-thyroglobulin (Tg) bind RHL-1 in vitro, and native Tg binds RHL-1 on the surface of PC C13 cells. After thyroid cells transformation, the surface expression of RHL-1 is inhibited in a measure that correlates with the mRNA and protein levels. Therefore, the RHL-1 inhibition at the mRNA, protein and plasma membrane expression follows a gradient that parallels the progressive acquisition of the fully transformed phenotype in the PC C13 system. The results reported in the present article, together with our previous data, suggest that RHL-1 expression could be regulated, at least in part, by the same transcription factors involved in the expression of the other molecules characteristic of the thyroid differentiated state.

The Rat Asialoglycoprotein Receptor Binds the Amino-Terminal Domain of Thyroglobulin

We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRDRHL-1) and the anti-rCRDRHL-1 antibody markedly inhibited 125I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRDRHL-1 was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRDRHL-1 on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRDRHL-1 specifically recognized a thyroglobulin fragment with an apparent Mr of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role.

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding sites at the carboxyl terminal extracellular domain.

Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 (RHL-1) oligomers

Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 (RHL-1) oligomers

A Specific Antibody to the Carbohydrate Recognition Domain of the Asialoglycoprotein Receptor RHL1 Subunit Does Not React with RHL2/3 but Blocks Ligand Binding

The rat asialoglycoprotein receptor (ASGPR) is believed to be a hetero-oligomer composed of three subunits, designated rat hepatic lectin 1, 2, and 3 (RHL1, 2, and 3). The carbohydrate recognition domains (CRDs) of RHL1 and RHL2/3 are 56% identical. We developed a polyclonal antibody that specifically recognizes the CRD of RHL1 but not RHL2/3. When purified ASGPRs were bound to ligand-Sepharose, the CRD of RHL1, but not RHL2 or RHL3, was resistant to digestion with subtilisin. Antibody against purified RHL1 CRD recognized only RHL1 in Western blot analysis of crude cell extracts or purified receptors without detectable cross-reaction to RHL2/3. Although it does not recognize the CRD of RHL2 or RHL3, this antibody specifically inhibited 80–90% of the cell surface or total cellular125I-ASOR binding to isolated rat hepatocytes and >90% of ligand binding to purified rat ASGPRs. The antibody also immunoprecipitates active ASGPRs containing all three RHL subunits. The results indicate that homo-oligomeric RHL2/3 complexes, able to bind ASOR, do not form on hepatocytes by subunit rearrangement.

Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 [correction of leptin-1] (RHL-1) oligomers.

The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p. b. following incubation with ferric ammonium citrate (<=100 microgram/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by approximately 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by approximately 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, beta-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.

Fatty Acylation of the Rat and Human Asialoglycoprotein Receptors: A CONSERVED CYTOPLASMIC CYSTEINE RESIDUE IS ACYLATED IN ALL RECEPTOR SUBUNITS

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.

Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor.

The rat liver asialoglycoprotein receptor consists of two types of subunits, a predominant polypeptide designated rat hepatic lectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comes in two differentially glycosylated forms. The exact stoichiometry and arrangement of the subunits in the RHL oligomer are not known. The carbohydrate-recognition domain of RHL-2/3 has been prepared by limited proteolysis of the liver receptor so that its properties can be compared with those of the corresponding domain of RHL-1 previously produced in a bacterial expression system. Binding studies indicate that while RHL-1 binds N-acetylgalactosamine with approximately 60-fold higher affinity than it binds galactose, RHL-2/3 has only 2-fold selectivity for N-acetylgalactosamine. In general, the pattern of monosaccharide-binding specificity for RHL-2/3 is similar to RHL-1, but the discrimination of various sugars relative to galactose is reduced substantially. Limited proteolysis and crosslinking studies demonstrate that RHL-2/3 is easily removed from the RHL oligomer in detergent solution and that RHL-1 remains at least trimeric following removal of RHL-2/3. These studies suggest that RHL-1 forms a ligand-binding core while RHL-2/3 acts more as an accessory subunit contributing to selective binding of certain oligosaccharide structures.

On the relationship between completion of N-acetyllactosamine oligosaccharide units and iodine content of thyroglobulin: a reinvestigation.

Lack of completion of N-acetyllactosamine-type glycosylation on thyroglobulin (Tg) has been implicitly considered as an etiological factor of some thyroid disorders, i.e. goiter and hypothyroidism. However, there is some evidence that Tg with incompletely processed N-acetyllactosamine glycans occurs in the normal gland. Recent findings demonstrated that exposed N-acetylglucosamine (GlcNAc) residues present on internalized glycoprotein in the thyrocyte may act as a retention signal that prevents lysosomal homing and triggers recycling of GlcNAc-bearing molecules through galactosyltransferase- and thyroperoxidase-containing compartments of the Golgi apparatus. This finding raises the possibilities 1) that exposed GlcNAc residues are not randomly distributed, but are mainly present on immature Tg; and 2) that this process promotes elongation of complex glycans, thereby eliminating the retention signal. To further validate this hypothesis, we reinvestigated the relationship between the iodine content and the glycan completion of porcine Tg of luminal origin. Tg subpopulations were separated according to their iodine content on rubidium chloride centrifugation gradients, and their interactions with various plant and animal lectins were analyzed in solid phase assays. Iodine content used as an index of age ranged from 0.6-1.2%. There was no significant correlation between iodine content and either neutral sugar or oligosaccharide content, as judged by chemical methods or interaction with [125I]Solanum tuberosum and [125I]Pisum sativum agglutinins. In contrast, the number of GlcNAc-accessible residues (as judged by interaction with [125I]Bandeiraea simplificolia II) decreased as iodine content increased. These changes were concomitant with an increase in galactose (measured by interaction with [125I]R-icinus communis and [125I]galactosidase (Gal)/GalNAc rat hepatic lectin) and sialic acid content. Related experiments using a Tg subpopulation depleted in GlcNAc-exposed residues by passage through a B. simplificolia II affinity column showed that the capacity of this subpopulation to bind to membranes was lowered compared to that of the total Tg. These results support the following conclusions: 1) in normal glands, all or part of the Tg molecules are secreted in an incompletely glycosylated form; and 2) iodine organification is correlated with glycan completion. Therefore, asialoagalactothyroglobulin appears to be a physiological precursor for an efficient recycling mediated by the GlcNAc receptor to the iodination site. New insights in thyroid disorders are discussed.

Fatty Acylation of the Rat Asialoglycoprotein Receptor: THE THREE SUBUNITS FROM ACTIVE RECEPTORS CONTAIN COVALENTLY BOUND PALMITATE AND STEARATE

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.

Inhibition of tyrosine phosphorylation in the rat hepatic lectin 1 subunit of the rat asialoglycoprotein receptor prevents ATP-dependent receptor inactivation in permeabilized hepatocytes

We previously reconstituted the ATP-dependent inactivation of asialoglycoprotein receptors (ASGPRs) in digitonin-permeabilized hepatocytes (Medh, J. D., and Weigel, P. H. (1991) J. Biol. Chem. 266, 8771-8778). Here we report that rat hepatic lectin 1 (RHL1) is the only ASGPR subunit that becomes radiolabeled when permeabilized washed hepatocytes are incubated at 4 degrees C in the presence of [gamma-32P]ATP; RHL2 and RHL3 are not radiolabeled. Phosphorylation of RHL1 was rapid (t1/2 appoximately 4 min) and complete within 30 min. Inclusion of 20 mM EDTA inhibited phosphorylation of RHL1 completely. Phosphoamino acid analysis identified Tyr(P) as the predominant (> 90%) radiolabeled phosphoamino acid. Addition of vanadate enhanced phosphorylation of Tyr in RHL1 4-fold. Phosphorylation of RHL1 occurred to the same extent in hepatocytes permeabilized with either 0.006% (w/v) or 0.055% digitonin and in the presence or the absence of ligand (50 micrograms/ml asialo-orosomucoid; ASOR) and/or 10 mM CaCl2. Sequential purification of active ASGPRs (using ASOR-Sepharose) and inactive ASGPRs from the ASOR-Sepharose flow-through (using anti-ASGPR antibody-Sepharose) demonstrated that radiolabeled RHL1 was present almost exclusively in active ASGPR oligomers. When permeabilized hepatocytes radiolabeled with [gamma-32P]ATP at 4 degrees C were warmed to 37 degrees C, a temperature at which ATP-dependent ASGPR inactivation occurs, RHL1 was dephosphorylated rapidly (t1/2 approximately 4 min) and completely within approximately 30 min. Western blot analysis using a monoclonal anti-Tyr(P) antibody showed that the steady-state level of endogenous Tyr(P) in RHL1 doubled as a result of ATP treatment at 4 degrees C and then decreased to undetectable levels upon warming to 37 degrees C. The protein-tyrosine kinase inhibitor tyrphostin 51 inhibited phosphorylation of RHL1 at 4 degrees C and also prevented ATP-dependent ASGPR inactivation at 37 degrees C. We conclude that phosphorylation of Tyr in RHL1 of active ASGPRs is a prerequisite for ATP-dependent ASGPR inactivation.

The rat hepatic lectin 1 subunit of the rat asialoglycoprotein receptor is a phosphoprotein and contains phosphotyrosine

The rat asialoglycoprotein receptor (ASGPR) is an integral transmembrane glycoprotein composed of three polypeptide subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. Each subunit contains one or more Ser and Thr residues in its cytoplasmic domain that are potential sites of phosphorylation; in addition, RHL1 also contains one cytoplasmic Tyr. Based on [32P]PO4 metabolic radiolabeling experiments, Takahashi et al. (Takahashi, T., Nakada, H., Okumura, T., Sawamura, T., and Tashiro, Y. (1985) Biochem. Biophys. Res. Commun. 126, 1054-1060) concluded that RHL2 and RHL3 are phosphoproteins but that RHL1 is not. We report here that RHL1 in active ASGPR is, in fact, a phosphoprotein. Western blot analysis using anti-Tyr(P) antibody identified Tyr(P) in RHL1 of affinity-purified ASGPRs. RHL2 and RHL3, which do not contain Tyr in their cytoplasmic domains, did not react with this antibody. When isolated hepatocytes were radiolabeled metabolically with [32P]PO4, RHL1, RHL2, and RHL3 became radiolabeled. Each ASGPR subunit was radiolabeled to a similar extent in the presence or absence of the ligand asialo-orosomucoid, indicating that functioning of the ASGPR does not change its steady-state 23P-radiolabeling. Phosphoamino acid analysis of radiolabeled ASGPR subunits identified Ser(P) as the predominant (approximately 95%) and Thr(P) as a minor (approximately 5%) phosphoamino acid in each polypeptide and confirmed the presence of Tyr(P) (approximately 1%) in RHL1. Furthermore, treatment of hepatocytes with 3 mM vandate at 37 degrees C for 30 min doubled the steady-state level of Tyr(P) in RHL1.

The major form of the murine asialoglycoprotein receptor: cDNA sequence and expression in liver, testis and epididymis.

Northern blot analysis of poly(A)+RNAs isolated from mouse liver or mouse testis (Te)/epididymis (Ep) reveals that both tissues express 1.5- and 7.5-kb transcripts which have extensive homology to the major form of the rat asialoglyco-protein receptor (ASGP-R). In situ hybridization studies have localized the expression of this ASGP-R-like transcript to late-stage sperm from Te and Ep of several different strains of mice. Swiss Webster mice express this ASGP-R-like transcript in late-stage spermatids at the time of release into the seminiferous tubule and in Ep sperm, while Balb/C, NIH Swiss and C57Bl/6 mice express this ASGP-R-like transcript predominantly in Ep sperm. cDNAs containing the entire coding region for this ASGP-R-like transcript have been cloned from mouse liver and mouse Te/Ep. These cDNAs are 100% identical in the coding region and 3'-untranslated region (UTR), but differ in the 5'-UTR. The gene encoding these cDNAs is called MHL-1, designating the major form of the mouse ASGP-R. The deduced amino acid (aa) sequence of MHL-1 shares 88% homology to the rat hepatic (He) lectin form 1 (RHL-1) and 78% homology to the human asialoglycoprotein receptor form 1 (H1). The three sites for N-linked glycosylation in the RHL-1 sequence are all conserved in the deduced MHL-1 sequence. Taken collectively, these data describe the cloning and sequencing of the MHL-1 cDNA and illustrate its deduced aa homology to RHL-1 and H1.(ABSTRACT TRUNCATED AT 250 WORDS)