Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.     

Novel Functional Complexity of Polycystin-1 by GPS Cleavage In Vivo: Role in Polycystic Kidney Disease

Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1^cFL) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1^deN) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1^U) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1^cFL and Pc1^deN traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1^deN is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1^deN trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis.     

Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif

The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.     

Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu downward arrow Ser(518) cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P(+1) cleavage site (Ser(518)) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P(-2) His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.     

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin

BACE1 (β-site β-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid β-peptide (Aβ) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of Aβ.     

Presenilins and gamma-secretase inhibitors affect intracellular trafficking and cell surface localization of the gamma-secretase complex components

The intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch is mediated by the presenilin (PS, PS1/PS2)-gamma-secretase complex, the components of which also include nicastrin, APH-1, and PEN-2. In addition to its essential role in gamma-secretase activity, we and others have reported that PS1 plays a role in intracellular trafficking of select membrane proteins including nicastrin. Here we examined the fate of PEN-2 in the absence of PS expression or gamma-secretase activity. We found that PEN-2 is retained in the endoplasmic reticulum and has a much shorter half-life in PS-deficient cells than in wild type cells, suggesting that PSs are required for maintaining the stability and proper subcellular trafficking of PEN-2. However, the function of PS in PEN-2 trafficking is distinct from its contribution to gamma-secretase activity because inhibition of gamma-secretase activity by gamma-secretase inhibitors did not affect the PEN-2 level or its egress from the endoplasmic reticulum. Instead, membrane-permeable gamma-secretase inhibitors, but not a membrane-impermeable derivative, markedly increased the cell surface levels of PS1 and PEN-2 without affecting that of nicastrin. In support of its role in PEN-2 trafficking, PS1 was also required for the gamma-secretase inhibitor-induced plasma membrane accumulation of PEN-2. We further showed that gamma-secretase inhibitors specifically accelerated the Golgi to the cell surface transport of PS1 and PEN-2. Taken together, we demonstrate an essential role for PSs in intracellular trafficking of the gamma-secretase components, and that selective gamma-secretase inhibitors differentially affect the trafficking of the gamma-secretase components, which may contribute to an inactivation of gamma-secretase.     

Polycystin-1 surface localization is stimulated by polycystin-2 and cleavage at the G protein-coupled receptor proteolytic site

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery.     

Biosynthesis of dystroglycan: processing of a precursor propeptide

Dystroglycan is a cytoskeleton-linked extracellular matrix receptor expressed in many cell types. Dystroglycan is composed of alpha- and beta-subunits which are encoded by a single mRNA. Using a heterologous mammalian expression system, we provide the first biochemical evidence of the alpha/beta-dystroglycan precursor propeptide prior to enzymatic cleavage. This 160 kDa dystroglycan propeptide is processed into alpha- and beta-dystroglycan (120 kDa and 43 kDa, respectively). We also demonstrate that the precursor propeptide is glycosylated and that blockade of asparagine-linked (N-linked) glycosylation did not prevent the cleavage of the dystroglycan precursor peptide. However, inhibition of N-linked glycosylation results in aberrant trafficking of the alpha- and beta-dystroglycan subunits to the plasma membrane. Thus, dystroglycan is synthesized as a precursor propeptide that is post-translationally cleaved and differentially glycosylated to yield alpha- and beta-dystroglycan.     

Pharmacological knock-down of the presenilin 1 heterodimer by a novel gamma -secretase inhibitor: implications for presenilin biology

Intramembranous cleavage of the beta-amyloid precursor protein by gamma-secretase is the final processing event generating amyloid-beta peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of gamma-secretase has been established. Here we describe for the first time that certain potent gamma-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel gamma-secretase inhibitor, [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/gamma-secretase complex assembly or trafficking. Furthermore, in cell-based systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the beta-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.     

Presenilin 1 regulates pharmacologically distinct gamma -secretase activities. Implications for the role of presenilin in gamma -secretase cleavage

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.     

Interaction of the amyloid precursor like protein 1 with the alpha2A-adrenergic receptor increases agonist-mediated inhibition of adenylate cyclase

Alpha2-adrenergic receptor agonists exert potent analgesic and sedative/hypnotic effects. In addition, they have been shown to be neuroprotective, but the mechanisms of these actions are still poorly defined. To isolate proteins that may control alpha2-adrenergic receptor function or trafficking, we performed a two-hybrid screen using the carboxy-terminal fourth intracellular tail of the alpha2A-adrenergic receptor as bait. This screen identified the amyloid precursor like protein 1 (APLP1), a homologue of the beta-amyloid precursor protein involved in Alzheimer's disease, as alpha2A-adrenergic receptor-binding protein. GST affinity chromatography revealed that APLP1 specifically interacts with all three human alpha2-adrenergic receptor subtypes and deletion mutant analysis confined the APLP1 domain involved in binding to alpha2-adrenergic receptors to the 13 amino acid residues Ser599-Ala611. Coimmunoprecipitations of transiently transfected cells with epitope-tagged APLP1 and alpha2-adrenergic receptors confirmed the interaction. Agonist treatment tended to increase the amount of alpha2A-adrenergic receptor associated with APLP1 while coimmunoprecipitations were not affected by the state of receptor phosphorylation or cotransfection of arrestin-3. Confocal laser microscopy showed that APLP1 causes a considerable shift of the alpha2A-adrenergic receptor localization from plasma membrane to intracellular compartments. Furthermore, cotransfection of alpha2A-adrenergic receptor and APLP1 into HEK293 cells significantly increased norepinephrine mediated inhibition of adenylate cyclase activity. These results suggest a possible role of APLP1 in regulation of alpha2A-adrenergic receptor trafficking. Moreover, we speculate that this interaction may present one mechanism by which alpha2-adrenergic receptor agonists exert their neuroprotective effects.     

SorCS3 does not require propeptide cleavage to bind nerve growth factor

The functional properties of the Vps10p-domain receptor SorCS3 are undescribed. Here, we examine its processing and sorting in cellular transfectants, and analyze the binding of potential ligands to the purified receptor. We show that SorCS3 is synthesized as a proprotein and converted to its mature form by N-terminal propeptide cleavage in distal Golgi compartments. The propeptide is not a requirement for normal processing of the receptor and does not prevent ligands from binding to the SorCS3 precursor form. Expression of wt and chimeric receptors further suggests that SorCS3 predominates on the plasma membrane, exhibits slow internalization and does not engage in intracellular trafficking. SorCS3 emerges as a new neurotrophin binding Vps10p-domain receptor functionally distinct from its relatives Sortilin and SorLA.     

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.     

Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1

Mucolipin-1 (ML1) is a member of the transient receptor potential ion channel superfamily that is thought to function in the biogenesis of lysosomes. Mutations in ML1 result in mucolipidosis type IV, a lysosomal storage disease characterized by the intracellular accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, and mucopolysaccharides. Little is known about how ML1 trafficking or activity is regulated. Here we have examined the processing and trafficking of ML1 in a variety of cell types. We find that a significant fraction of ML1 undergoes cell type-independent cleavage within the first extracellular loop of the protein during a late step in its biosynthetic delivery. To determine the trafficking route of ML1, we systematically examined the effect of ablating adaptor protein complexes on the localization of this protein. Whereas ML1 trafficking was not apparently affected in fibroblasts from mocha mice that lack functional adaptor protein complex (AP)-3, small interfering RNA-mediated knockdown revealed a requirement for AP-1 in Golgi export of ML1. Knockdown of functional AP-2 had no effect on ML1 localization. Interestingly, cleavage of ML1 was not compromised in AP-1-deficient cells, suggesting that proteolysis occurs in a prelysosomal compartment, possibly the trans-Golgi network. Our results suggest that posttranslational processing of ML1 is more complex than previously described and that this protein is delivered to lysosomes primarily via an AP-1-dependent route that does not involve passage via the cell surface.     

Dystroglycan is associated to the disulfide isomerase ERp57

Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG-ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.     

The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.     

COPI-mediated retrograde transport is required for efficient gamma-secretase cleavage of the amyloid precursor protein

Sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of beta-amyloid peptide, which is a key determinant in Alzheimer's disease. Since several putative locations for gamma-secretase cleavage have been identified along the secretory pathway, trafficking of APP may be of importance for beta-amyloid peptide production. Here we have studied the role of retrograde transport in APP processing. We found that APP interacts with the beta subunit of the coatomer protein I (COPI) complex, which is involved in retrograde transport. In line with a role of retrograde trafficking in APP transport, inhibition of COPI-dependent transport altered APP trafficking, decreased APP cell surface expression, and coincided with a profound reduction in gamma-secretase cleavage. These results suggest that COPI-dependent retrograde transport is important for APP processing and influences production of beta-amyloid peptide.