Biomedical subjects

Jean-Marc Navenot

Publications and source records attributed to Jean-Marc Navenot.

8 recordsLinked to original sources

PubMed2006

Structure-activity relationship study on small peptidic GPR54 agonists.

Metastin (kisspeptin-54) is an endogenous ligand that modulates gonadotropin-releasing hormone (GnRH) secretion through the interaction with a G protein-coupled receptor (GPCR), GPR54. The short-chain C-terminal decapeptide amide, metastin (45-54) (kisspeptin-10), exerts the identical bioactivities to metastin, such as metastasis suppression of cancer cells and inhibition of trophoblast migration and invasion. In order to understand the structural requirement for GPR54 agonistic activity, structure-activity relationship (SAR) study on pentapeptide-based C-terminal metastin analogues was carried out. As a result, H-Amb-Nal(2)-Gly-Leu-Arg-Trp-NH2 34 was identified as a novel GPR54 agonist that possessed the most potent GPR54 agonistic activity reported so far.

Alkylation↗

PubMed2006

Characterization of constitutively active mutants of G protein-coupled receptors.

The ability of G protein-coupled receptors to transduce signaling typically is induced by the binding of an appropriate ligand (agonist), resulting in a conformational change of the receptor and the subsequent interaction with the G protein heterotrimer. Some mutants of G protein-coupled receptors, known as constitutively active mutants, have the capacity to activate the G protein-signaling cascade even in the absence of ligand. In this chapter, we describe three methods that most directly allow characterization of constitutively active mutants and discriminate them from the wild-type receptors. All methods are based on the spontaneous signaling function in the absence of ligand and its consequences on the receptor.

Animals↗

PubMed2005

Kisspeptin-10-induced signaling of GPR54 negatively regulates chemotactic responses mediated by CXCR4: a potential mechanism for the metastasis suppressor activity of kisspeptins.

The product of the KiSS-1 gene is absent or expressed at low level in metastatic melanoma and breast cancer compared with their nonmetastatic counterparts. A polypeptide derived from the KiSS-1 product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Galphaq subunits (GPR54 or KiSS-1R). To study the mechanism by which Kp-10 antagonizes metastatic spread, the effect on CXCR4-mediated signaling, which has been shown to direct organ-specific migration of tumor cells, was determined. Kp-10 blocked chemotaxis of tumor cells expressing CXCR4 in response to low and high concentrations of SDF-1/CXCL12 and inhibited mobilization of calcium ions induced by this ligand. Pretreatment with Kp-10 did not induce down-modulation of cell surface CXCR4 expression, reduce affinity for SDF-1/CXCL12, or alter Galphai subunit activation stimulated by this ligand. Although Kp-10 stimulated prolonged phosphorylation of extracellular signal-regulated kinase 1/2, it inhibited the phosphorylation of Akt induced by SDF-1. The ability of Kp-10 to inhibit signaling and chemotaxis induced by SDF-1 indicates that activation of GPR54 signaling may negatively regulate the role of CXCR4 in programming tumor metastasis.

Animals↗

PubMed2005

Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity.

Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45-54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45-54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.

Amino Acid Motifs↗

PubMed2003

Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2.

CCR5 is a G protein-coupled receptor for RANTES, MIP-1alpha, MIP-1beta, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.

Amino Acid Motifs↗

PubMed2003

S-protein is expressed in necrotic fibers in Duchenne muscular dystrophy and polymyositis.

Complement regulatory proteins (CD55, CD59) and a fluid-phase complement regulator (S-protein) expression and distribution were studied in Duchenne muscular dystrophy (DMD) and polymyositis by Western blots and immunocytochemistry. In muscle samples from control subjects, no specific signal was detected for CD55 or S-protein, and CD59 was present on the sarcolemma of the muscle fibers. In DMD and polymyositis, Western blots demonstrated a 18-20 kDa band corresponding to CD59, as well as a signal corresponding to S-protein. Immunocytochemistry showed a colocalization between complement membrane attack complex (MAC), a molecule previously demonstrated in necrotic muscle fibers in DMD and polymyositis, and S-protein in necrotic fibers of DMD and polymyositis. Necrotic muscle fibers were more numerous in muscle biopsies of DMD patients with stronger signals for S-protein in Western blots. These results suggest that S-protein is not able to prevent the full assembly of MAC in necrotic fibers of patients with DMD and polymyositis, but might instead inactivate MAC deposits present inside necrotic fibers or participate in the clearance of MAC-attacked muscle fibers.

Antibodies, Monoclonal↗

PubMed2002

Role of CD4 hinge region in GP120 utilization by immunoglobulin domain 1.

Immunoglobulin-like domain 1 of CD4 (D1-CD4) promotes HIV infection by binding the envelope glycoprotein (ENV) and exposing its coreceptor-binding site. To study CD4-ENV-coreceptor interactions, we characterized hybrid receptors having domains 1 and 2 of CD4 (D1D2-CD4) joined to the N-terminus of chemokine receptors CCR5, CXCR4, CXCR2, and DARC. Hybrid receptors showed conserved ENV-coreceptor specificity in cell-cell fusion assays. Although D1D2-CD4-CCR5 was sufficient to permit ENV-mediated fusion, D1-CD4-CCR5 and human D1/mouse D2-CD4-CCR5 lacked CD4 function and binding to a neutralizing antibody mapped to D1-CD4. Chimeric D1D2-CD4 joined to CCR5 revealed that the C-terminal 20 residues of human D2-CD4 are required for efficient ENV-mediated fusion. Mutagenesis of hybrid receptors showed the importance of residues forming D1-D2 CD4 interdomain contacts and hinge region proximal residues. Mutagenesis of WT human CD4 confirmed that residues forming D1-D2 interdomain contacts and hinge-region proximal residues contribute positively to CD4 activity in the full-length receptor.

Amino Acid Sequence↗

PubMed2002

A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists.

CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.

Amino Acid Substitution↗