Low density lipoprotein receptor-related protein 1 couples β1 integrin activation to degradation

Low density lipoprotein receptor-related protein (LRP) 1 modulates cell adhesion and motility under normal and pathological conditions. Previous studies documented that LRP1 binds several integrin receptors and mediates their trafficking to the cell surface and endocytosis. However, the mechanism by which LRP1 may regulate integrin activation remains unknown. Here we report that LRP1 promotes the activation and subsequent degradation of β1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface β1 integrin, binds the integrin activator kindlin2 and stimulates β1 integrin–kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, β1 integrin activation and cell adhesion. Interestingly, a loss of LRP1 induces the accumulation of several integrin receptors on the cell surface. Following internalization, intracellular trafficking of integrins is driven by LRP1 in a protein kinase C- and class II myosin-dependent manner. Ultimately, LRP1 dictates the fate of endocytosed β1 integrin by directing it down the pathway of lysosomal and proteasomal degradation. We propose that LRP1 mediates cell adhesion by orchestrating a multi-protein pathway to activate, traffic and degrade integrins. Thus, LRP1 may serve as a focal point in the integrin quality control system to ensure a firm connection to the extracellular matrix.     

Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.     

Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E _max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E _max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.     

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.     

Kank proteins: structure, functions and diseases

The Kank family of proteins, Kank1–Kank4, are characterized by their unique structure, coiled-coil motifs in the N-terminal region, and ankyrin-repeats in the C-terminal region, with an additional motif, the KN motif, at the N-terminus. Kank1 was obtained by positional cloning of a tumor suppressor gene in renal cell carcinoma, while the other members were found by homology search. The family is involved in the regulation of actin polymerization and cell motility through signaling pathways containing PI3K/Akt and/or unidentified modulators/effectors. Their relationship to diseases such as cancer, and to neuronal and developmental disorders, will be an important subject of future study.     

Effect of prenatal and neonatal pantothenic acid deficiency on rat intestinal phosphatases

Alkaline phosphatase activity was increased in the distal part of the small intestine of pantothenic acid deficient neonatal rats, while acid phosphatase activity was slightly increased and protein concentration was decreased throughout the small intestine. The growth and maturation of the distal part of the small intestine were retarded more severely than in the proximal part.     

Effect of prenatal and neonatal pantothenic acid deficiency on rat intestinal phosphatases

Summary Alkaline phosphatase activity was increased in the distal part of the small intestine of pantothenic acid deficient neonatal rats, while acid phosphatase activity was slightly increased and protein concentration was decreased throughout the small intestine. The growth and maturation of the distal part of the small intestine were retarded more severely than in the proximal part.     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

LDL receptor relatives at the crossroad of endocytosis and signaling

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.     

Nitric oxide mediates histamine induced down-regulation of H<Subscript>2</Subscript> receptor mRNA and internalization of the receptor protein (R1)

During agonist-dependent long-term stimulation of cells, histamine receptor subtypes are frequently down-regulated. However, the mechanisms underlying the modulation of receptor expression during long-term histamine stimulation have yet to be resolved. Based on our recently reported results showing an H₁-mediated down-regulation of histamine H₂ receptor mRNA in endothelial cells, our aim was to characterize the mechanism controlling rapid and long-term histamine-mediated modulation of H₂ receptor expression in more detail. We were able to show that the histamine-induced down-regulation of H₂ receptor mRNA and cell surface expression lasting for 24 h was accompanied by augmentation of the receptor protein level in the cytoplasmatic fraction of endothelial cells for this time period. Furthermore, changes in receptor protein levels in whole-cell lysate were negligible, indicating that the rapid and prolonged modulation of cell surface H₂ receptor levels by histamine was regulated solely via internalization. The role of nitric oxide (NO) as a key mediator in histamine-stimulated cell responses was underlined by subsequent studies showing the attenuation of histamine-induced H₂ receptor mRNA down-regulation and protein trafficking following NO synthase isozyme inhibition.Received 11 March 2003; received after revision 11 June 2003; accepted 17 June 2003     

The odd couple: signal transduction and endocytosis

Cell surface receptors are used to transmit extracellular information. The activation of cell surface receptors initiates signal transduction and receptor endocytosis. Signal transduction and the endosomal transport of activated receptors require precise regulation. New concepts for the integration of endocytosis and signaling arise from recent findings that suggest bidirectional interplay of these two processes. This review discusses the following questions: (i) do activated cell surface receptors modify the endosomal system to promote internalization and endosomal traffic, and (ii) do internalized cell surface receptors use specifically localized signaling complexes to generate specific biological signals?     

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.     

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.     

Dual Vα T cells

The assumption that T cells can only express a single receptor for antigen has in recent years been shown to be incorrect. However, the finding that a substantial number of T cells express two distinct antigen receptors at the cell surface raises a number of questions. In particular, it has been suggested that cells expressing low levels of a self-reactive T cell receptor may escape self-tolerance mechanisms and in certain situations trigger the onset of autoimmune disease. Such a hypothesis in turn raises questions central to the understanding of the nature of T cell recognition and the process of thymocyte maturation.     

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation.

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T' parallel order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T' parallel for a 3-h period at 37 degrees C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.     

The complexity of parathyroid hormone-related proteinsignalling

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.Received 19 June 2003; received after revision 10 July 2003; accepted 21 July 2003     

AID and Apobec3G haphazard deamination and mutational diversity

Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C → U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell “mutators”. Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides “surrogate” insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.