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1.
FASEB J ; 26(5): 2060-71, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22291440

RESUMO

Vasoactive intestinal peptide (VIP) plays a major role in pathophysiology. Our previous studies demonstrated that the VIP sequence 6-28 interacts with the N-terminal ectodomain (N-ted) of its receptor, VPAC1. Probes for VIP and receptor antagonist PG97-269 were synthesized with a photolabile residue/Bpa at various positions and used to explore spatial proximity with VPAC1. PG97-269 probes with Bpa at position 0, 6, and 24 behaved as high-affinity receptor antagonists (K(i)=12, 9, and 7 nM, respectively). Photolabeling experiments revealed that the [Bpa(0)]-VIP probe was in physical contact with VPAC1 Q(135), while [Bpa(0)]-PG97-269 was covalently bound to G(62) residue of N-ted, indicating different binding sites. In contrast, photolabeling with [Bpa(6)]- and [Bpa(24)]-PG97-269 showed that the distal domains of PG97-269 interacted with N-ted, as we previously showed for VIP. Substitution with alanine of the K(143), T(144), and T(147) residues located in the first transmembrane domain of VPAC1 induced a loss of receptor affinity (IC(50)=1035, 874, and 2070 nM, respectively), and pharmacological studies using VIP2-28 indicated that these three residues play an important role in VPAC1 interaction with the first histidine residue of VIP. These data demonstrate that VIP and PG97-269 bind to distinct domains of VPAC1.


Assuntos
Peptídeos/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Peptídeos/química , Marcadores de Fotoafinidade , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Homologia de Sequência de Aminoácidos
2.
Cancer Res ; 71(9): 3341-51, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21415167

RESUMO

Resistance to apoptosis is a recurrent theme in colon cancer. We have shown previously that the 7-transmembrane spanning receptor OX1R for orexins promotes robust apoptosis in the human colon cancer cell line HT29 through an entirely novel mechanism involving phosphorylation of tyrosine-based motifs in OX1R. Here, we investigated the status of OX1R in a large series of human colorectal tumors and hepatic metastases. All primary colorectal tumors regardless of their localization and Duke's stages and all hepatic metastases tested expressed OX1R mRNA and/or protein. In sharp contrast, adjacent normal colonocytes or hepatocytes as well as control normal tissues were negative. Next, we showed that nine human colon cancer cell lines established from primary tumors or metastases expressed OX1R mRNA and underwent important apoptosis on orexin-A challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly used drug in colon cancer chemotherapy, 5-fluorouracil. When human colon cancer cells were xenografted in nude mice, orexin-A administered at day 0 strongly slowed the tumor growth and even reversed the development of established tumors when administered 7 days after cell inoculation. Orexin-A also acts by promoting tumor apoptosis in vivo because caspase-3 is activated in tumors on orexin treatment of nude mice. These findings support that OX1R is an Achilles heel of colon cancers, even after metastasis or chemoresistance. They suggest that OX1R agonists might be novel candidates for colon cancer therapy.


Assuntos
Apoptose/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Receptores de Neuropeptídeos/biossíntese , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Células HCT116 , Células HT29 , Humanos , Neoplasias Hepáticas/genética , Receptores de Orexina , Orexinas , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética
3.
FASEB J ; 23(12): 4069-80, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19661287

RESUMO

The orexin neuropeptides promote robust apoptosis in cancer cells. We have recently shown that the 7-transmembrane-spanning orexin receptor OX1R mediates apoptosis through an original mechanism. OX1R is equipped with a tyrosine-based inhibitory motif ITIM, which is tyrosine-phosphorylated on receptor activation, allowing the recruitment and activation of the tyrosine phosphatase SHP-2, leading to apoptosis. We show here that another motif, immunoreceptor tyrosine-based switch motif (ITSM), is present in OX1R and is mandatory for OX1R-mediated apoptosis. This conclusion is based on the following observations: 1) a canonical ITSM sequence is present in the first intracellular loop of OX1R; 2) mutation of Y(83) to F within ITSM abolished OX1R-mediated apoptosis but did not alter orexin-induced inositol phosphate formation or calcium transient via coupling of OX1R to G(q) protein; 3) mutation of Y(83) to F further abolished orexin-induced tyrosine phosphorylation in ITSM and subsequent recruitment of SHP-2 by the receptor. Finally, we developed a structural model of OX1R showing that the spatial localization of phosphotyrosines in ITSM and ITIM in OX1R is compatible with their interaction with the two SH2 domains of SHP-2. These data represent the first evidence for a functional role of an ITSM in a 7-transmembrane-spanning receptor.


Assuntos
Apoptose/fisiologia , Membrana Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Membrana Celular/química , Cricetinae , Cricetulus , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Neuropeptídeos/metabolismo , Receptores de Orexina , Orexinas , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Proteínas Recombinantes
4.
J Mol Neurosci ; 36(1-3): 249-53, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18592417

RESUMO

Vasoactive intestinal peptide (VIP) is implicated in many physiological and pathophysiological processes, and its receptors are promising targets for the development of new drugs. The human VPAC1 receptor for VIP and pituitary adenylate cyclase-activating polypeptide is a class II G protein coupled receptor. The N-terminal ectodomain (N-ted) of the VPAC1 receptor is a major VIP binding site. To determinate the high resolution structure of the VPAC1 receptor N-ted, large quantities of purified recombinant N-ted produced are required. The N-ted sequence (31-144), which is fused to thioredoxin protein and 6xHis tag, was expressed into Origami Escherichia coli strain. Purification of recombinant N-ted using Ni-NTA affinity column associated to Nu-polyacrylamide gel electrophoresis analysis reveals the presence of one single band of Mw 19,000 corresponding to the purified recombinant N-ted. The purified N-ted was able to recognize VIP and the selective antagonist PG96-269. About 5-10 mg of functional purified protein/liter of bacterial culture is currently produced. This is a crucial step to determine the structure of functional human VPAC1 receptor N-ted by nuclear magnetic resonance.


Assuntos
Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/química , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/isolamento & purificação , Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Conformação Proteica , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/genética
5.
FASEB J ; 22(6): 1993-2002, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18198212

RESUMO

Orexins acting at the G protein-coupled receptor (GPCR) OX1R have recently been shown to promote dramatic apoptosis in cancer cells. We report here that orexin-induced apoptosis is driven by an immunoreceptor tyrosine-based inhibitory motif (ITIM) (IIY(358)NFL) present in the OX1R. This effect is mediated by SHP-2 phosphatase recruitment via a mechanism that requires Gq protein but is independent of phospholipase C activation. This is based on the following observations: 1) mutation of Y(358) into F abolished orexin-induced tyrosine phosphorylation in ITIM, orexin-induced apoptosis, and uncoupled OX1R from Gq protein in transfected Chinese hamster ovary (CHO) cells; 2) orexin-induced apoptosis in CHO cells expressing recombinant OX1R and in colon cancer cells expressing the native receptor was abolished by treatment with the tyrosine phosphatase inhibitor PAO and by transfection with a dominant-negative mutant of SHP-2; 3) orexins were unable to promote apoptosis in fibroblast cells invalidated for the G alpha q subunit and transfected with OX1R cDNA, whereas they promoted apoptosis in cells equipped with G alpha q and OX1R; and 4) the phospholipase C inhibitor U-73122 blocked orexin-stimulated inositol phosphate formation, whereas it had no effect on orexin-induced apoptosis in CHO cells expressing OX1R. These data unravel a novel mechanism, whereby ITIM-expressing GPCRs may trigger apoptosis.


Assuntos
Motivos de Aminoácidos/fisiologia , Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neuropeptídeos/fisiologia , Receptores Acoplados a Proteínas G/química , Receptores de Neuropeptídeos/química , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Orexina , Orexinas , Receptores Acoplados a Proteínas G/genética , Receptores Imunológicos , Receptores de Neuropeptídeos/genética , Transfecção , Fosfolipases Tipo C/metabolismo
6.
Regul Pept ; 123(1-3): 181-5, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15518910

RESUMO

The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1-30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1-30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.


Assuntos
Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/genética , Animais , Ligação Competitiva , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Peptídeo Intestinal Vasoativo/metabolismo
7.
J Biol Chem ; 279(44): 45875-86, 2004 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-15310763

RESUMO

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/farmacologia , Receptores de Neuropeptídeos/fisiologia , Animais , Células CHO , Caspases/fisiologia , Divisão Celular , Cricetinae , Citocromos c/metabolismo , Células HT29 , Humanos , Receptores de Orexina , Orexinas , Receptores Acoplados a Proteínas G
8.
Mol Pharmacol ; 64(6): 1565-74, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14645688

RESUMO

The VPAC1 receptor for vasoactive intestinal peptide (VIP) belongs to the class II family of G protein-coupled receptors and is coupled to Gs protein/adenylyl cyclase. We assessed whether 10 different Ser/Thr residues in human VPAC1 receptor intracellular domains play a role in the process of VIP-induced desensitization/internalization by performing a site-directed mutagenesis study. The Ser/Thr residues mutated to Ala include potential G protein-coupled receptor kinase, protein kinase A and protein kinase C targets that are of particular interest for VPAC1 receptor desensitization. The data show that when Chinese hamster ovary cells expressing wild-type receptors were pretreated for 5 min with VIP (50 nM), receptor desensitization occurred with a 10-fold right shift of the ED50 for adenylyl cyclase activation. When the construct with the widest span of mutations was studied, there was no longer any short-term desensitization. By using constructs with fewer and fewer mutations, we identified Ser447 in the C-terminal tail to be crucial for rapid desensitization. We also showed that Ser447 plays an essential role for VIP-induced VPAC1 phosphorylation in Chinese hamster ovary cells. Furthermore, we demonstrated that none of the mutated Ser/Thr residues was involved in down-regulation after a 12-h treatment of cells with 50 nM VIP. Neither were they involved in VIP and VIP-induced receptor internalization as shown using a novel fluorescein-tagged VIP and VPAC1 receptor bearing a Flag epitope in the N-terminal domain and a green fluorescent protein at the C terminus. We conclude that Ser447, a likely G protein-coupled receptor kinase target, is crucial for VIP-induced phosphorylation and rapid desensitization of VPAC1 receptor.


Assuntos
Fragmentos de Peptídeos/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/agonistas , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Serina/metabolismo , Sequência de Aminoácidos/genética , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína/genética , Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Serina/química , Serina/genética , Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia
9.
J Biol Chem ; 278(27): 24759-66, 2003 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-12690118

RESUMO

The VPAC1 receptor mediates the action of two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide. It is a class II G protein-coupled receptor-activating adenylyl cyclase (AC). The role of the N-terminal extracellular domain of hVPAC1 receptor for VIP binding is now established (Laburthe, M., Couvineau, A. and Marie, J. C. (2002) Recept. Channels 8, 137-153), but nothing is known regarding the cytoplasmic domains responsible for AC activation. Here, we constructed a large series of mutants by substituting amino acids with alanine in the intracellular loops (IL) 1, 2, and 3 and proximal C-terminal tail of the receptor. The mutation of 40 amino acids followed by expression of mutants in chinese hamster ovary cells showed the following. (i) Mutations IL1 result in the absence of expression of mutants, suggesting a role of this loop in receptor folding. (ii) All residues of IL2 can be mutated without alteration of receptor expression and AC response to VIP. (iii) Mutation of residues IL3 points to the specific role of lysine 322 in the efficacy of the stimulation of AC activity by VIP. This efficacy is reduced by 50% in the K322A mutant. (iv) The proximal C-terminal tail is equipped with another important amino acid since mutation of glutamic acid 394 reduces AC response by 50%. The double mutant K322A/E394A exhibits a drastic reduction of >85% in the efficacy of VIP in stimulating AC activity in membranes and cAMP response in intact cells without alteration of receptor expression or affinity for VIP. These data highlight the role of charged residues in IL3 and the proximal C-terminal tail of hVPAC1 receptor for agonist-induced AC activation. Because these charged residues are absolutely conserved in class II receptors for peptides, which are all mediating AC activation, they may play a general role in coupling of class II receptors with the Gs protein.


Assuntos
Adenilil Ciclases/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Adenilil Ciclases/genética , Animais , Células CHO , Cricetinae , Ativação Enzimática/genética , Humanos , Mutação , Estrutura Terciária de Proteína/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Transdução de Sinais/genética , Relação Estrutura-Atividade
10.
J Biol Chem ; 277(40): 37016-22, 2002 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-12133828

RESUMO

The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC(50) for secretin/EC(50) for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123-144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln(135) Thr(136) and Gly(140) Ser(141) in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor.


Assuntos
AMP Cíclico/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Secretina/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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