Juxtamembrane Segment Research Articles

Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor.

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two-subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH-TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I-labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N-linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N-linked glycan. Elimination by mutagenesis of the most strategic N-linked glycan (LHR-N317Q) generated only a trace amount of LHR cleavage. Removal of the other N-linked glycans had no additive effect. A much greater degree of cleavage ( approximately 50%) was evident in a chimeric LH-TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317-367) was replaced with the corresponding domain of the TSHR (residues 363-418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR-NET317-319 replaced with TSHR-GQE367-369, or by substitution of the same three amino-acid residues with AAA (LHR-NET317-319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino-acid specificity in a region important for TSHR cleavage (GQE367-369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.

Identification of a Juxtamembrane Segment of the Glutamate Receptor δ2 Subunit Required for the Plasma Membrane Localization

Glutamate receptor δ2 subunit (GluRδ2) is selectively expressed in cerebellar Purkinje cells and is specifically targeted to parallel fiber synapses, where GluRδ2 plays important roles in synaptic plasticity, motor learning and synapse formation. Here, we investigated the mechanism of GluRδ2 sorting using MDCK epithelial cells. Immunocytochemical analysis of subcellular localization showed that GluRδ2 expressed in MDCK cells was predominantly distributed in the plasma membrane. By examining the subcellular localization of GluRδ2 mutants with various deletions in the carboxyl-terminal cytoplasmic region, we identified J segment adjacent to transmembrane segment M4 as a key region for the efficient targeting of GluRδ2 to the plasma membrane. On the other hand, the carboxyl terminus of GluRδ2 essential for interaction with PDZ domain proteins was dispensable for the plasma membrane localization. Our results suggest that the juxtamembrane segment and the PDZ domain target site at the carboxyl terminus of GluRδ2 play differential roles in plasma membrane targeting, synapse localization and signal transduction.

Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors.

Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

Novel Recognition Motif on Fibroblast Growth Factor Receptor Mediates Direct Association and Activation of SNT Adapter Proteins

Fibroblast growth factors (FGFs) stimulate tyrosine phosphorylation of a membrane-anchored adapter protein, FRS2/SNT-1, promoting its association with Shp-2 tyrosine phosphatase and upstream activators of Ras. Using the yeast two-hybrid protein-protein interaction assay, we show that FRS2/SNT-1 and a newly isolated SNT-2 protein directly bind to FGF receptor-1 (FGFR-1). A juxtamembrane segment of FGFR-1 and the phosphotyrosine-binding domain of SNTs are both necessary and sufficient for interaction in yeast and in vitro, and FGFR-mediated SNT tyrosine phosphorylation in vivo requires these segments of receptor and SNT. Our findings establish SNTs as direct protein links between FGFR-1 and multiple downstream pathways. The SNT binding motif of FGFR-1 is distinct from previously described phosphotyrosine-binding domain recognition motifs, lacking both tyrosine and asparagine residues.

Identification and characterization of RPTP ρ, a novel RPTP μ/ κ-like receptor protein tyrosine phosphatase whose expression is restricted to the central nervous system

We describe the cloning, chromosomal localization and characterization of RPTP ρ, a new member of the RPTP μ/ κ phosphatase subfamily. Receptor tyrosine phosphatases in this subfamily are comprised of a MAM domain near the N-terminal, an immunoglobulin-like domain, four fibronectin type III repeats, a single transmembrane domain, and a large juxtamembrane segment followed by two intracellular phosphatase domains. An alternatively spliced mini-exon was identified in the extracellular segment of RPTP ρ, between the fourth fibronectin type III repeat and the transmembrane domain. The RPTR ρ gene was mapped to human chromosome 20 and mouse chromosome 2. Northern blot analysis demonstrated that RPTP ρ expression was restricted to the central nervous system, and in situ hybridization studies showed that the RPTP ρ transcript was distributed throughout the murine brain and spinal cord. Exceptionally high levels of the transcript were present in the cortex and olfactory bulbs during perinatal development, but were down-regulated during postnatal week two. The motifs found in the extracellular segment of type II receptor protein tyrosine phosphatases are commonly found in neural cell adhesion molecules, suggesting that RPTP ρ may be involved in both signal transduction and cellular adhesion in the central nervous system.

Analysis of Cell-Cycle Profiles in Transfected Cells Using a Membrane-Targeted GFP

Analysis of Cell-Cycle Profiles in Transfected Cells Using a Membrane-Targeted GFP

Mutational Analysis of the L1 Neuronal Cell Adhesion Molecule Identifies Membrane-Proximal Amino Acids of the Cytoplasmic Domain That Are Required for Cytoskeletal Anchorage

The preferential localization of the L1 cell adhesion molecule in the axons and growth cones of differentiating neurons suggests the existence of a mechanism for targeting or anchoring the molecule to these locations. We have used B28 glioma cells, which have an extremely flattened morphology, as a model system to study the organization of L1 on the cell surface. Transfection of L1 cDNA into B28 cells results in expression of the L1 protein in organized linear cell surface arrays which are codistributed with cytoskeletal stress fibers, but not with microtubules or intermediate filaments. Transfection studies with L1 deletion mutants identify the juxtamembrane segment of the cytoplasmic domain as the critical entity for arrangement of L1 into ordered cell surface arrays. The seventh cytoplasmic amino acid of L1, lysine 1150, and to a lesser extent the fourth cytoplasmic amino acid, lysine 1147, appear to be critical residues for maintaining normal L1 anchorage and distribution.

Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo.

The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine-phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.

A tyrosine residue in the juxtamembrane segment of the platelet-derived growth factor beta-receptor is critical for ligand-mediated endocytosis.

The importance of tyrosine residues in ligand-mediated endocytosis of the platelet-derived growth factor beta-receptor was analyzed using a series of tyrosine residue-mutated beta-receptors, which together cover all of the tyrosine residues in the juxtamembrane segment, the kinase insert, and the carboxyl-terminal tail; also certain of the tyrosine residues within the first and second parts of the kinase domain were examined. Of all of these tyrosine residues, only Tyr-579 seemed to be important for internalization, since mutation of this residue resulted in substantial reduction in the rate of ligand-induced receptor internalization (approximately 60% of the wild-type level). Replacement of Tyr-579 by either an aromatic (Phe) or a nonaromatic (Asp) residue reduced the efficiency of the mutant receptors in internalization to the same extent, suggesting that the role of Tyr-579 in the beta-receptor is different from that of the previously described tyrosine-based internalization motifs, which were first determined for the low density lipoprotein receptor. Tyr-579 has been found to be an autophosphorylation site in the beta-receptor. Moreover, the internalization rate of a kinase negative receptor mutant was not altered by the additional mutation of Tyr-579. Thus, it is likely that phosphorylation of Tyr-579 is important for ligand-induced internalization of the beta-receptor.

Two steps of insulin receptor internalization depend on different domains of the beta-subunit.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases.

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human platelet-derived growth factor (PDGF) beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF beta-receptor and downstream components in the signal transduction pathway.

Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.

Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.