The COOH terminus of megalin regulates gene expression in opossum kidney proximal tubule cells

We recently reported that megalin is subjected to regulated intramembrane proteolysis (RIP) and includes 1) protein kinase C (PKC)-regulated, metalloprotease-mediated ectodomain shedding producing a membrane-bound megalin COOH-terminal fragment (MCTF) and 2) gamma-secretase-mediated cleavage of the MCTF producing a soluble megalin intracellular domain (MICD). Based on studies of RIP of other receptors, the MICD is predicted to target to the nucleus and regulate gene expression. To determine whether RIP of megalin regulates proximal tubule gene expression, we stably expressed the transfected MCTF (tMCTF) or transfected MICD (tMICD) in opossum kidney proximal tubule (OKP) cells and examined the resulting phenotype. Immunoblotting and immunocytochemical analysis of tMCTF cells showed the tMCTF was expressed and constitutively processed by gamma-secretase. Analysis of specific protein expression in tMCTF- and tMICD-transfected cells using Western blot showed endogenous megalin and Na(+)/H(+) exchanger 3 (NHE3) protein expression to be dramatically lower than that of control cells. Expression of other proteins including myosin VI, beta-adaptin, and the Na-K-ATPase appeared unchanged. Analysis of specific mRNA expression using quantitative real-time PCR showed megalin and NHE3 mRNA levels were significantly lower in tMCTF- and tMICD-transfected cells compared with controls. Inhibition of gamma-secretase activity in tMCTF cells resulted in an 8- to 10-fold recovery of megalin mRNA within 4 h. These data show that the COOH-terminal domain of megalin regulates expression of specific proteins in OKP cells and provides the first evidence that RIP of megalin may be part of a signaling pathway linking protein absorption and gene expression in proximal tubule.     

A disintegrin and metalloproteinase (ADAM)-mediated ectodomain shedding of ADAM10

A disintegrin and metalloproteinase (ADAM) 10 is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a range of proteins including the amyloid precursor protein implicated in Alzheimer's disease. In this study we demonstrate that ADAM10 itself is subject to shedding by one or more ADAMs. Expression of epitope-tagged wild-type ADAM10 in SH-SY5Y cells enabled the detection of a soluble ectodomain in conditioned medium. Shedding of the ADAM10 ectodomain was inhibited by a known ADAM inhibitor with a reciprocal accumulation of the full-length mature protein in both cell lysates and extracellular membrane vesicles. Shedding was also stimulated by phorbol ester treatment of cells. A glycosylphosphatidylinositol-anchored form of ADAM10 lacking the cytosolic, transmembrane and α-helical juxtamembrane regions of the wild-type protein was shed in a similar manner. Furthermore, a truncated soluble ADAM10 construct, although correctly post-translationally processed and catalytically active against a synthetic peptide substrate, was incapable of shedding cell-associated amyloid precursor protein. Finally, we show that ADAM9 is, at least in part, responsible for the ectodomain shedding of ADAM10. In conclusion, this is a new mechanism by which levels of ADAM10 are regulated and may have implications in a range of human diseases including Alzheimer's disease.     

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.     

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.     

Aldosterone stimulates activity and surface expression of NHE3 in human primary proximal tubule epithelial cells (RPTEC).

The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na(+) channel (ENaC), the Na(+)-K(+)-ATPase and the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na(+)- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pH(i)- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.     

Specific functions of Drosophila amyloid precursor-like protein in the development of nervous system and nonneural tissues

Drosophila amyloid precursor-like protein (APPL) is expressed extensively in the nervous system soon after neuronal differentiation. By utilizing different transgenic flies, we studied the physiological function of two APPL protein forms, membrane-bound form (mAPPL) and secreted form (sAPPL), in neural development. We found that neither deletion nor overexpression of APPL protein altered the gross structure of mushroom bodies in the adult brain. No changes were detected in cell types and their relative ration in embryo-derived cultures from all APPL mutants. However, the neurite length was significantly increased in mutants overexpressing mAPPL. In addition, mutants lacking sAPPL had numerous neurite branches with abnormal lamellate membrane structures (LMSs) and blebs, while no apoptosis was detected in these neurons. The abnormal neurite morphology was most likely due to the disorganization of the cytoskeleton, as shown by double staining of actin filaments and microtubules. Electrophysiologically, A-type K+ current was significantly enhanced, and spontaneous excitatory postsynaptic potentials (sEPSPs) were greatly increased in APPL mutants lacking sAPPL. Moreover, panneural overexpression of different forms of APPL protein generated different defects of wings and cuticle in adult flies. Taken together, our results suggest that both mAPPL and sAPPL play essential roles in the development of the central nervous system and nonneural tissues.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

Prostaglandin E2 modulates proximal tubule Na-ATPase activity: Cooperative effect between protein kinase A and protein kinase C

Previous data showed that prostaglandin E₂ (PGE₂) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na⁺-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE₂ on PT Na⁺-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE₂ on PT Na⁺-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE₂ increased [³⁵S]GTPγS binding; (2) GDPβS abolished the inhibitory effect of PGE₂; (3) PGE₂ increased PKA activity; (4) the inhibitory effect of PGE₂ was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE₂ on PT Na⁺-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE₂ stimulates Ca²⁺-independent phospholipase A₂ activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE₂ on proximal tubule sodium reabsorption.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

Internalization of exogenously added memapsin 2 (beta-secretase) ectodomain by cells is mediated by amyloid precursor protein

Memapsin 2 (beta-secretase) is the protease that initiates cleavage of amyloid precursor protein (APP) leading to the production of amyloid-beta (Abeta) peptide and the onset of Alzheimer's disease. Both APP and memapsin 2 are Type I transmembrane proteins and are endocytosed into endosomes where APP is cleaved by memapsin 2. Separate endocytic signals are located in the cytosolic domains of these proteins. We demonstrate here that the addition of the ectodomain of memapsin 2 (M2(ED)) to cells transfected with native APP or APP Swedish mutant (APPsw) resulted in the internalization of M2(ED) into endosomes with increased Abeta production. These effects were reduced by treatment with glycosylphosphatidylinositol-specific phospholipase C. The nontransfected parental cells had little internalization of M2(ED). The internalization of M2(ED) was dependent on the endocytosis signal in APP, because the expression of a mutant APP that lacks its endocytosis signal failed to support M2(ED) internalization. These results suggest that exogenously added M2(ED) interacts with the ectodomain of APP on the cell surface leading to the internalization of M2(ED), supported by fluorescence resonance energy transfer experiments. The interactions between the two proteins is not due to the binding of substrate APPsw to the active site of memapsin 2, because neither a potent active site binding inhibitor of memapsin 2 nor an antibody directed to the beta-secretase site of APPsw had an effect on the uptake of M2(ED). In addition, full-length memapsin 2 and APP, immunoprecipitated together from cell lysates, suggested that the interaction of these two proteins is part of the native cellular processes.     

The amyloid precursor-like protein 2 and the adenoviral E3/19K protein both bind to a conformational site on H-2Kd and regulate H-2Kd expression

A protein of unknown physiological function, called amyloid precursor-like protein 2 (APLP2), forms an association with the murine class I molecule K(d) that is up-regulated by the presence of the adenoviral protein E3/19K. We have extended these findings to show that APLP2 and E3/19K associate preferentially with folded K(d) and not with the open form. APLP2 was detectable at the cell surface, but its surface expression was not up-regulated by the concurrent expression of K(d). Experimental down-regulation of APLP2 expression caused a consistent increase in the surface expression of K(d), indicating that APLP2 normally reduces K(d) surface expression. These data suggest a role for APLP2 in controlling the maturation of major histocompatibility complex class I molecules.     

Stimulation of the proximal tubule Na+-ATPase activity by adenosine A(2A) receptor

The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.     

UNC-71, a disintegrin and metalloprotease (ADAM) protein,regulates motor axon guidance and sex myoblast migration in C. elegans

The migration of cells and growth cones is a process that is guided by extracellular cues and requires the controlled remodeling of the extracellular matrix along the migratory path. The ADAM proteins are important regulators of cellular adhesion and recognition because they can combine regulated proteolysis with modulation of cell adhesion. We report that the C. elegans gene unc-71 encodes a unique ADAM with an inactive metalloprotease domain. Loss-of-function mutations in unc-71 cause distinct defects in motor axon guidance and sex myoblast migration. Many unc-71 mutations affect the disintegrin and the cysteine-rich domains, supporting a major function of unc-71 in cell adhesion. UNC-71 appears to be expressed in a selected set of cells. Genetic mosaic analysis and tissue-specific expression studies indicate that unc-71 acts in a cell non-autonomous manner for both motor axon guidance and sex myoblast migration. Finally, double mutant analysis of unc-71 with other axon guidance signaling molecules suggests that UNC-71 probably functions in a combinatorial manner with integrins and UNC-6/netrin to provide distinct axon guidance cues at specific choice points for motoneurons.     

The role of ADAM10 and ADAM17 in the ectodomain shedding of angiotensin converting enzyme and the amyloid precursor protein.

Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-alpha converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the alpha-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP.