Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor.
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ABSTRACT:
SUBMITTER: Rosorius O
PROVIDER: S-EPMC1134189 | biostudies-other | 1993 Jun
REPOSITORIES: biostudies-other
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Project description:The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) mediates the intracellular transport of newly synthesized lysosomal enzymes containing mannose 6-phosphate on their N-linked oligosaccharides. In addition to its role in lysosome biogenesis, the CI-MPR interacts with a number of different extracellular ligands at the cell surface, including latent transforming growth factor-beta, insulin-like growth factor-II, plasminogen, and urokinase-type plasminogen activator receptor (uPAR), to regulate cell growth and motility. We have solved the crystal structure of the N-terminal 432 residues of the CI-MPR at 1.8 A resolution, which encompass three out of the 15 repetitive domains of its extracytoplasmic region. The three domains, which exhibit similar topology to each other and to the 46 kDa cation-dependent mannose 6-phosphate receptor, assemble into a compact structure with the uPAR/plasminogen and the carbohydrate-binding sites situated on opposite faces of the molecule. Knowledge of the arrangement of these three domains has allowed us to propose a model of the entire extracytoplasmic region of the CI-MPR that provides a context with which to envision the numerous binding interactions carried out by this multi-faceted receptor.
| S-EPMC424385 | biostudies-literature
Project description:The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.
| S-EPMC1218697 | biostudies-other
Project description:The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting approximately 60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5-9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1-3, interacts with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes.
| S-EPMC2787381 | biostudies-literature
Project description:BackgroundThe mannose 6-phosphate receptor homology (MRH) domain-containing family of proteins, which include recycling receptors (mannose 6-phosphate receptors, MPRs), resident endoplasmic reticulum (ER) proteins (glucosidase II β-subunit, XTP3-B, OS-9), and a Golgi glycosyltransferase (GlcNAc-phosphotransferase γ-subunit), are characterized by the presence of one or more MRH domains. Many MRH domains act as lectins and bind specific phosphorylated (MPRs) or non-phosphorylated (glucosidase II β-subunit, XTP3-B and OS-9) high mannose-type N-glycans. The MPRs are the only proteins known to bind mannose 6-phosphate (Man-6-P) residues via their MRH domains.Scope of reviewRecent biochemical and structural studies that have provided valuable insight into the glycan specificity and mechanisms of carbohydrate recognition by this diverse group of MRH domain-containing proteins are highlighted.Major conclusionsCurrently, three-dimensional structures are known for ten MRH domains, revealing the conservation of a similar fold. OS-9 and the MPRs use the same four residues (Gln, Arg, Glu, and Tyr) to bind mannose.General significanceThe MRH domain-containing proteins play key roles in the secretory pathway: glucosidase II, XTP3-B, and OS-9 are involved in the recognition of nascent glycoproteins, whereas the MPRs play an essential role in lysosome biogenesis by targeting Man-6-P-containing lysosomal enzymes to the lysosome.
| S-EPMC3150748 | biostudies-literature
Project description:The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ? 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 ?M, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.
| S-EPMC4410830 | biostudies-literature
Project description:DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.
| S-EPMC2526700 | biostudies-literature
Project description:In eukaryotic cells, post-translational modification of secreted proteins and intracellular protein transport between organelles are ubiquitous features. One of the most studied systems is the N-linked glycosylation pathway in the synthesis of secreted glycoproteins (Schrag et al. 2003). The N-linked glycoproteins are subjected to diverse modifications and are transported through ER and Golgi apparatus to their final destinations in- and outside the cell. Incorporation of cargo glycoproteins into transport vesicles is mediated by transmembrane cargo receptors, which have been identified as intracellular lectins. For example, mannose 6-phosphate receptors (Ghosh et al. 2003) function as a cargo receptor for lysosomal proteins in the trans-Golgi network, whereas ERGIC-53 (Zhang et al. 2003) and its yeast orthologs Emp46/47p (Sato and Nakano 2002) are transport lectins for glycoproteins that are transported out of ER.
| S-EPMC7121444 | biostudies-literature
Project description:The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.
| S-EPMC2707234 | biostudies-literature
Project description:Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR-/- MPS VII mice. The availability of both MR+/+ and MR-/- mice allowed us to study the effects of eliminating the MR on MR- and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human beta-glucuronidase (GUS). In MR+/+ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg/kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P- and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPR-targeted ERT.
| S-EPMC1622795 | biostudies-literature
Project description:BackgroundRyanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation.ResultsHere we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis.ConclusionsThe crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.
| S-EPMC6784350 | biostudies-literature