Paige Holst

GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF

We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

GFRalpha-2 and GFRalpha-3 are two new receptors for ligands of the GDNF family.

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRα-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRα-1 or GFRα-2, a novel receptor related to GFRα-1. Both GFRα-1 and GFRα-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRα-1 or GFRα-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRα-1 bind GDNF more efficiently than neurturin, while cells expressing GFRα-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRα-1 is the primary receptor for GDNF and GFRα-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRα receptor family. This putative receptor, designated GFRα-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRα-1 and GFRα-2. Analysis of the tissue distribution of GFRα-1, GFRα-2, GFRα-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRαs and Ret are expressed in many of the same tissues, suggesting that GFRαs mediate the action of GDNF family ligands in vivo.

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: Effects of acidic amino acid substitutions

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40 → Asp substitution.