Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions

Equilibrium binding of 125I-nerve growth factor (125I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75NTR), cells coexpressing both receptors where p75NTR is occupied, and cells expressing only p75NTR, revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12nnr5 cells suggested that liganded and unliganded p75NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75NTR. These data are consistent with receptor allosterism, and prompted a search for TrkA/p75NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound 125I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.     

Non-lysosomal cycling pathway for atrial natriuretic peptide activated by protein kinase C in human NPE cells

Atrial natriuretic peptide (ANP) stimulates aqueous humor formation in primates, but the membrane-bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [125I]ANP bound to natriuretic peptide C receptors was characterized in fetal human nonpigmented ciliary epithelial (NPE) cells. [125I]ANP which bound to cells at 4 degreesC was detected in the cell interior after a temperature shift to 37 degreesC. Appearance of ligand within the cell peaked at 5 min, and then declined towards zero over 20 min. The endocytosis inhibitor phenylarsine oxide blocked the appearance of internalized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloroquine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of internalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoylglycerol but not by 4alpha phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol ester-stimulated loss of internalized ligand occurred in the presence of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [125I]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the medium, and phorbol esters stimulated its rate of appearance by approximately 2 fold. A similar stimulation occurred when cells containing internalized ligand, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE cells is endocytosed, and that protein kinase C activates a non-lysosomal pathway for ANP retroendocytosis in these cells.     

High affinity nerve growth factor binding displays a faster rate of association than p140trk binding. Implications for multi-subunit polypeptide receptors

Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.     

Solubilization of nerve growth factor receptors of rabbit superior cervical ganglia

Nerve growth factor (NGF) receptors of rabbit superior cervical ganglia can be solubilized by treatment with detergents and readily assayed in the soluble state. Triton X-100 and deoxycholate reduce specific binding of NGF to ganglia membranes. In membranes treated with Triton X-100 (0.5 to 2.0%) the reduction in NGF binding by membranes is accompanied by a corresponding increase in binding in the supernatant fluid. NGF binding in soluble preparations can be rapidly assayed by precipitating NGF bound to receptors with polyethylene glycol under conditions in which unbound NGF is not precipitated. NGF binding to soluble preparations is saturable whether evaluated by the binding of 125I-NGF or by diluting 125I-NGF with native NGF. Using both techniques, the dissociation constant for NGF binding to soluble receptors is about 0.2 nM, the same as its dissociation constant from receptor sites in intact membranes. NGF binding to soluble receptors displays a high degree of peptide specificity, similar to receptor sites in intact membranes of superior cervical ganglia. A method of labeling NGF with 125I-3(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester is described which leads to binding properties that are superior to those obtained with previously described 125I-NGF preparations.     

Cytoplasmic domain of E-selectin contains a non-tyrosine endocytosis signal

E-selectin is an activation-dependent, endothelial cell-restricted adhesion molecule that is internalized and degraded rapidly once expressed on the cell surface. Tyrosine-containing structural motifs play an important role in the internalization of a number of integral proteins, and the membrane-proximal E-selectin cytoplasmic tyrosine residue (Tyr582) conforms to the endocytosis motif proposed previously. To determine the endocytosis motif in E-selectin, we selectively introduced truncation, substitution, and deletion mutations to the cytoplasmic tail of E-selectin. We analyzed the internalization kinetics of surface-expressed wild-type and mutant E-selectin constructs in transiently transfected Chinese hamster ovary cells using 125I-labeled E-selectin monoclonal antibody (125I-P6E2) in an acid elution assay. Interestingly, truncation immediately membrane proximal to Tyr582 (DeltaDGS construct) did not alter internalization kinetics significantly (DeltaDGS versus wild-type, mean surface half-life = 42 versus 45 min, respectively). Thus, it appears that the tyrosine residues are not required for internalization of E-selectin. Additional analyses indicated that Ser581 was necessary but alone was insufficient for surface E-selectin endocytosis. Thus, we conclude that there exists a novel non-tyrosine-containing endocytosis signal in the cytoplasmic tail which involves Ser581 and residues membrane-proximal to it.     

The dependence of the endocytosis of the epidermal growth factor receptors on the degree of receptor occupancy

Two subpopulations of epidermal growth factor (EGF) receptor with different affinity to EGF have been demonstrated for many cell types. There are reasons to assume a key role of high-affinity receptors in stimulation of cell response to EGF. Nevertheless, characteristics of its action remain obscure. In the present work an attempt has been made to study internalization and intracellular compartmentalization of high-affinity receptors. For this purpose endocytosis of 125I-EGF in A431 cells was stimulated by low (less than 1 nM) EGF concentrations as well as after blocking of low-affinity binding sites with specific monoclonal antibodies 2E9. By subcellular fractionation in 17% Percoll gradient it was demonstrated that in both cases internalized 125I-EGF was found first in light, and then in heavy endosomes staying there for a long time. Effectiveness of 125I-EGF delivery to prelysosomal heavy endosomes as well as initial internalization rate is maximal at low EGF concentrations and is reduced dramatically with increasing of receptor occupancy. Monoclonal antibody Mab108 specifically recognizing high-affinity receptors were capable of stimulation of receptor internalization with initial rate higher than that of high EGF concentrations, but Mab108-receptor complexes were localized in light endosomes. Preincubation of the cells with low concentrations of EGF led to redistribution of considerable portion of 125I-Mab108 into heavy endosomes, which may be a result of high-affinity receptor tyrosine kinase activation. Our data confirm a hypothesis of TK involvement in regulation of both internalization and sorting of EGF-receptor complexes. Structural organization of high-affinity receptors is not sufficient for receptor targeting to degradation pathway.     

Saturation of the endocytic pathway for the transferrin receptor does not affect the endocytosis of the epidermal growth factor receptor

Cell-surface receptors that undergo clathrin-mediated endocytosis contain short amino acid sequences in their cytoplasmic domain that serve as internalization signals. Interactions between these sequences and components of the endocytic machinery should become limiting upon overexpression of the constitutively recycling transferrin receptor (TfR). A tetracycline-responsive system was used to induce overexpression of the TfR up to 20-fold in HeLa cells. Internalization assays indicate the rate of 125I-transferrin uptake per surface TfR is reduced by a factor of 4 in induced cells. Consistent with endocytosis being the rate-limiting step, TfRs shift from an endosomal to more of a plasma membrane distribution with TfR overexpression. The clathrin-associated protein AP-2 has been proposed to interact directly with the cytoplasmic domain of many receptors, yet no changes in the amount or distribution of AP-2 were detected in induced cells. The internalization rate for the epidermal growth factor receptor was also measured, with or without induction of TfR expression. Even though endocytosis of the TfR is saturated in induced cells, 125I-labeled epidermal growth factor continues to be internalized at a rate identical to that seen in uninduced cells. We propose that there are different limiting steps for the endocytosis of these two receptors.     

Molecular characterization of the signal responsible for the targeting of the interleukin 2 receptor beta chain toward intracellular degradation

During receptor-mediated endocytosis, most growth factor receptors are transported to late endocytic compartments and degraded. This process is important to control their expression on the cell surface and requires sorting in early endocytic compartments. Little is known about the mechanisms and the signals involved. We have studied the signal involved in targeting the interleukin 2 receptor beta chain (IL2Rbeta), a member of the cytokine receptor superfamily, toward degradation after internalization. We show that a motif of 8 amino acids in the cytosolic tail of IL2Rbeta is sufficient to target a normally recycling receptor toward degradation. Deletion of this signal strongly impairs IL2Rbeta degradation. Further molecular characterization of the motif shows that it does not resemble the well documented tyrosine and dileucine families of trafficking signals.     

Functional expression of TrkA receptors in hippocampal neurons

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.     

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF)

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.     

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy

In addition to its role as a survival factor, nerve growth factor (NGF) has been implicated in initiating apoptosis in restricted cell types both during development and after terminal cell differentiation. NGF binds to the TrkA tyrosine kinase and the p75 neurotrophin receptor, a member of the tumor necrosis factor cytokine family. To understand the mechanisms underlying survival versus death decisions, the TrkA receptor was introduced into oligodendrocyte cell cultures that undergo apoptosis in a p75-dependent manner. Here we report that activation of the TrkA NGF receptor in oligodendrocytes negates cell death by the p75 receptor. TrkA-mediated rescue from apoptosis correlated with mitogen-activated protein kinase activation. Concurrently, activation of TrkA in oligodendrocytes resulted in suppression of c-jun kinase activity initiated by p75, whereas induction of NFkappaB activity by p75 was unaffected. These results indicate that TrkA-mediated rescue involves not only activation of survival signals but also simultaneous suppression of a death signal by p75. The selective interplay between tyrosine kinase and cytokine receptors provides a novel mechanism that achieves alternative cellular responses by merging signals from different ligand-receptor systems.     

p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

Urokinase-type plasminogen activator receptor is internalized by different mechanisms in polarized and nonpolarized Madin-Darby canine kidney epithelial cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin-Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0 degreesC, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5-10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

Delta and kappa opioid receptors are differentially regulated by dynamin-dependent endocytosis when activated by the same alkaloid agonist

Many alkaloid drugs used as analgesics activate multiple opioid receptors. Mechanisms that distinguish the actions of these drugs on the regulation of individual micro, delta, and kappa receptors are not understood. We have observed that individual cloned opioid receptors differ significantly in their regulation by rapid endocytosis in the presence of alkaloid drug etorphine, a potent agonist of mu, delta, and kappa opioid receptors. Internalization of epitope-tagged delta opioid receptors from the plasma membrane is detectable within 10 min in the presence of etorphine. In contrast, kappa receptors expressed in the same cells remain in the plasma membrane and are not internalized for >/=60 min, even when cells are exposed to saturating concentrations of etorphine. The rapid internalization of delta receptors is specifically inhibited in cells expressing K44E mutant dynamin I, suggesting that type-specific internalization of opioid receptors is mediated by clathrin-coated pits. Examination of a series of chimeric mutant kappa/delta receptors indicates that at least two receptor domains, including the highly divergent carboxyl-terminal cytoplasmic tail, determine the type specificity of this endocytic mechanism. We conclude that structurally homologous opioid receptors are differentially sorted by clathrin-mediated endocytosis following activation by the same agonist ligand. These studies identify a fundamental mechanism of receptor regulation mediating type-specific effects of analgesic drugs that activate more than one type of opioid receptor.     

The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56(Lck) and p59(Fyn). Studies of mutated TCR and chimeric CD4-CD3gamma molecules demonstrated that CD3gamma did not contain a recycling signal in itself. In contrast, the only sorting information in CD3gamma was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3gamma molecules. Finally, we found a correlation between the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed.     

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.     

IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life

We have tested the role of glial cell line-derived neurotrophic factor (GDNF) in regulating a group of putatively nociceptive dorsal root ganglion (DRG) neurons that do not express calcitonin gene-related peptide (CGRP) and that downregulate the nerve growth factor (NGF) receptor tyrosine kinase, TrkA, after birth. We show that mRNA and protein for the GDNF receptor tyrosine kinase, Ret, are expressed in the DRG in patterns that differ markedly from those of any of the neurotrophin receptors. Most strikingly, a population of small neurons initiates expression of Ret between embryonic day 15.5 and postnatal day 7.5 and maintains Ret expression into adulthood. These Ret-expressing small neurons are selectively labeled by the lectin IB4 and project to lamina IIi of the dorsal horn. Ret-expressing neurons also express the glycosyl-phosphatidyl inositol-linked (GPI-linked) GDNF binding component GDNFR-alpha and retrogradely transport 125I-GDNF, indicating the presence of a biologically active GDNF receptor complex. In vitro, GDNF supports the survival of small neurons that express Ret and bind IB4 while failing to support the survival of neurons expressing TrkA and CGRP. Together, our findings suggest that IB4-binding neurons switch from dependence on NGF in embryonic life to dependence on GDNF in postnatal life and are likely regulated by GDNF in maturity.     

Endocytosis and recycling of neurokinin 1 receptors in enteric neurons

Neurotransmission depends on the availability of transmitter and on the presence of functional, high-affinity receptors at the plasma membrane that are capable of binding ligand. The pathway, mechanism and function of endocytosis and recycling of the substance P or neurokinin 1 receptor in enteric neurons were studied using fluorescent substance P, receptor antibodies and confocal microscopy. In both the soma and neurites, substance P induced rapid, clathrin-mediated internalization of the neurokinin 1 receptor into early endosomes, which also contained the transferrin receptor. After 4-8 h, there was a return in surface neurokinin 1 receptor immunoreactivity in the soma, which was not prevented by cycloheximide, and was thus independent of new protein synthesis. This return was prevented by acidotropic agents, therefore required endosomal acidification. This suggests that the neurokinin 1 receptor recycles in the soma. In contrast, in neurites, substance P and the neurokinin 1 receptor remained in endosomes and recycling was not detected. Neurons of the myenteric plexus were heavily innervated by substance P-containing nerve fibers, and K(+)-stimulated release of endogenous substance P from cultured neurons induced internalization of the neurokinin 1-receptor. Therefore, endogenous substance P may induce endocytosis of the neurokinin 1 receptor. In the soma, endocytosis and recycling correlated with loss and recovery of functional binding sites for substance P. suggesting that this process contributes to the regulation of peptidergic neurotransmission. Thus, ligand-induced endocytosis of the neurokinin 1 receptor in myenteric neurons is associated with a loss of surface receptors and functional binding sites. Since release of endogenous substance P induces neurokinin 1 receptor internalization, and neurokinin 1 receptor neurons are innervated by substance P-containing fibers, endocytosis of neuropeptide receptors may regulate neurotransmission.     

Polar redistribution of 125I-labelled insulin on the plasma membrane of cultured human lymphocytes

Cultured human lymphocytes of the IM-9 line have specific binding sites for 125I-insulin that have been characterized in detail. When the hormone--cell interaction was studied by quantitative electron microscopic (EM) autoradiography, it was shown that initial binding was to the plasma membrane and that at 37 degrees C a small fraction of the ligand was internalized by the cell. The internalized ligand was found preferentially localized in lysosomes in the Golgi region of the cell. It is well known that di- or multivalent ligands redistribute in the plane of the membrane before internalization. We now report that following initial binding the univalent ligand insulin also undergoes a polar redistribution ('capping') before internalization.     

Quantitative-topographic and temporal characterization of myoelectrical activation patterns: new diagnostic possibilities in neurology, physiotherapy and orthopaedics

We previously reported that Chinese hamster ovary (CHO) cell lines overexpressing mutated human insulin receptors (hIRs) in which the tyrosine residues 1162 and 1163 were replaced by phenylalanines (CHO-Y2) exhibited a marked defect in hormone-induced receptor internalization as compared to CHO transfectants overexpressing wild-type hIRs (CHO-R). These two cell lines are now used to compare the role of tyrosines 1162-1163 in basal and ligand-stimulated receptor internalization as well as in receptor turnover. We show here that (1) in CHO-Y2 cells, basal endocytosis, like insulin-induced internalization, was markedly altered despite normal receptor turnover and (2) in both CHO-R and CHO-Y2 cells, basal receptor endocytosis was altered by tunicamycin, an inhibitor of protein N-glycosylation, whereas insulin-induced internalization was not. These results support a role for tyrosines 1162-1163 of the IR beta-subunit major autophosphorylation domain in both basal and ligand-stimulated receptor endocytosis and provide evidence that the two processes follow distinct pathways.