Recycling and resensitization of the neurokinin 1 receptor: influence of agonist concentration and Rab GTPases

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4,-6 and-7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed RMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K-d 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Methylene spacer-regulated structural variation in cobalt(II/III) complexes with bridging acetate and Salen- or Salpn-type Schiff-base ligands

Two linear, trinuclear mixed-valence complexes, [Co-II{(mu-L-1)(mu-OAc)Co-III (OAc)}(2)] (1) and [Co-II(mu-L-2) (mu-OAc)Co-III(OAc)}(2)] (2) and two mononuclear Con' complexes [Co-III{L-3)(OAc)] (3), and [Co-III {L-4}(OAc)] (4) were prepared and the molecular structures of 1, 2 and 4 elucidated on the basis of X-ray crystallography [OAc = Acetate ion, H2L1 = H(2)Salen 1,6-bis(2-hydroxyphenyl)-2,5-diazahexa-1,5-diene, H2L2 H2Me2-Salen = 2,7-bis(2-hydroxyphenyl)-2,6-diazaocta-2,6-diene, H2L3 = H(2)Salpn = 1,7-bis(2-hydroxyphenyl)-2,6-diazahepta1,6-diene, H2L4 = H(2)Me(2)Salpn = 2,8-bis(2-hydroxyphenyl)3,7-diazanona-2,7-dienel. In complexes I and 2, the acetate groups show both monodentate and bridging bidentate coordination modes, whereas chelating bidentate acetate is present in 4. The terminal (CoN2O4)-N-III centres in 1 and 2 exhibit uniform facial arrangements of both non-bridged N2O and bridging O-3 donor sets and the Co-II centre is coordinated to six (four phenoxo and two acetato) oxygen atoms of the bridging ligands. The effective magnetic moment at room temperature corresponds to the presence of high-spin Coll in both 1 and 2. The complexes 1 and 2 are thus Co-III(S = 0)Co-II(S = 3/2)-Co-II(S = 0) trimers. Complexes 3 and 4 are monomeric and diamagnetic containing low-spin Co-III(S = 0) with chelating tetradentate Schiff base and bidentate acetate. Calculations based on DFT rationalise the formation of trinuclear or monomiclear complexes. (C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008).

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Characterisation of an s-type low molecular weight glutenin subunit of wheat and its proline and glutamine-rich repetitive domain

The low molecular weight glutenin subunits (LMW-GS) are major components of the glutenin polymers which determine the elastomeric properties of wheat (Triticum aestivum L.) gluten and dough. They comprise a complex mixture of components and have proved to be difficult to purify for detailed characterisation. The mature LMW subunit proteins comprise two structural domains, with one domain consisting of repeated sequences based on short peptide motifs. DNA sequences encoding this domain and a whole subunit were expressed in Escherichia coli and the recombinant proteins purified. Detailed comparisons by spectroscopy (CD, FT-IR) and dynamic light scattering indicated that the repetitive and non-repetitive domains of the proteins formed different structures with the former having an extended conformation with an equilibrium between poly-L-proline II-like structure and type II’ b-turns, and the latter a more compact globular structure rich in a-helix. Although the structures of these two domains appear to form independently, dynamic light scattering of the whole subunit dissolved in trifluoroethanol(TFE) suggested that they interact, leading to a more compact conformation. These observations may have relevance to the role of the LMW-GS in gluten structure and functionality.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Amphioxus type I keratin cDNA and the evolution of intermediate filament genes

We report the cloning of an intermediate filament (IF) cDNA from the cephalochordate amphioxus that encodes a protein assignable to the type I keratin group. This is the first type I keratin reported from an invertebrate. Molecular phylogenetic analyses reveal that amphioxus also possesses a type II keratin, and that the genes encoding short-rod IF proteins underwent different patterns of duplication in vertebrates and their closest relatives, the cephalochordates. Extensive IF gene duplication and divergence may have facilitated the origin of new specialised cell types in vertebrates.

Endothelin-converting enzyme 1 degrades neuropeptides in endosomes to control receptor recycling

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.