GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Neurotrophic factors and their receptors

Four GDNF ligands (GDNF, neurturin, artemin and persephin), and mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF) protect midbrain dopaminergic neurons that degenerate in Parkinson's disease. Each GDNF ligand binds a specific coreceptor GDNF family receptor α (GFRα), leading to the formation of a heterotetramer complex, which then interacts with receptor tyrosine kinase RET, the signalling receptor. The present thesis describes the structural and biochemical characterization of the GDNF2-GFRα12 complex and the MANF and CDNF proteins. Previous and current mutation data and comparison between GDNF-GFRα1 and artemin-GFRα3 binding interfaces show that N162GFRα1, I175GFRα1, V230GFRα1, Y120GDNF and L114GDNF are the specificity determinants among different ligand-coreceptor pairs. The structure suggests that sucrose octasulphate, a heparin mimic, interacts with a region R190-K202 within domain 2 of GFRα1. Mutating these residues on the GFRα1 surface, which are not in the GDNF binding region, affected RET phosphorylation, which provides a putative RET binding region in domain 2 and 3 of GFRα1. The structural comparison of the GDNF-GFRα1 and artemin-GFRα3 complexes shows a difference in bend angle between the ligand monomers. This variation in bend angle of the ligand may affect the kinetics of RET phosphorylation. To confirm that the difference is not due to crystallization artefacts, I crystallized the GDNF-GFRα1 complex without SOS in different cell dimensions. The structure of the second GDNF-GFRα1 complex is very similar to the previous one, suggesting that the difference between the artemin-GFRα3 and GDNF-GFRα1 complexes are intrinsic, not due to crystal packing. Finally, MANF and CDNF are bifunctional proteins with extracellular neurotrophic activity and ER resident cytoprotective role. The crystal structures of MANF and CDNF are presented here. Intriguingly, the structures of both the neurotrophic factors do not show structural similarity to any of previously known growth factor superfamilies; instead they are similar to saposins, the lipid-binding proteins. The N-terminal domain of MANF and CDNF contain conserved lysines and arginines on its surface, which may interact with negatively charged head groups of phospholipids, as saposins do. Thus MANF and CDNF may provide neurotrophic activities by interacting with a lipo-receptor. The structure of MANF shows a CXXC motif forming internal disulphide bridge in the natively unfolded C-terminus. This motif is common to reductases and disulphide isomerases. It is thus tempting to speculate that the CXXC motif of MANF and CDNF may be involved in oxidative protein folding, which may explain its cytoprotective role in the ER.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

Electric Field–Enhanced Sensitivity of Grafted Ligands and Receptors

BACKGROUND: Particle-based agglutination tests consisting of receptors grafted to colloidal microparticles are useful for detecting small quantities of corresponding ligands of interest in fluid test samples, but detection limits of such tests are limited to a certain concentration and it is most desirable to lower the detection limits and to enhance the rate of recognition of ligands. METHODS: A mixture of receptor-coated colloidal microparticles and corresponding ligand was sandwiched between 2 indium tin oxide-coated glass plates. Electrohydrodynamic drag from an alternating-current electric field applied perpendicular to the plates increased the local concentration of the colloidal particles, improving the chances of ligand-receptor interaction and leading to the aggregation of the colloidal particles. RESULTS: With this technique the sensitivity of the ligand-receptor recognition was increased by a factor as large as 50. CONCLUSIONS: This method can improve the sensitivity of particle-based agglutination tests used in immuno-assays and many other applications such as immunoprecipitation and chemical, sniffing. (C) 2007 American Association for Clinical Chemistry.

VEGF-mediated cell survival in non-small-cell lung cancer: implications for epigenetic targeting of VEGF receptors as a therapeutic approach

Aims: To evaluate the potential therapeutic utility of histone deacetylase inhibitors (HDACi) in targeting VEGF receptors in non-small-cell lung cancer. Materials & methods: Non-small-cell lung cancer cells were screened for the VEGF receptors at the mRNA and protein levels, while cellular responses to various HDACi were examined. Results: Significant effects on the regulation of the VEGF receptors were observed in response to HDACi. These were associated with decreased secretion of VEGF, decreased cellular proliferation and increased apoptosis which could not be rescued by addition of exogenous recombinant VEGF. Direct remodeling of the VEGFR1 and VEGFR2 promoters was observed. In contrast, HDACi treatments resulted in significant downregulation of the Neuropilin receptors. Conclusion: Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for lung cancer patients in the clinical setting.

Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac β 1–4 Gal as the binding epitope with GalNac β 1–3 Gal and GlcNac β 1–3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac β 1–4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces.