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.

Understanding Human Drug Conjugating Enzymes; Regio- and Stereoselectivity in Sulfotransferase 1A3 and the UDP-Glucuronosyltransferases

Sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs) are important detoxification enzymes and they contribute to bioavailability and elimination of many drugs. SULT1A3 is an extrahepatic enzyme responsible for the sulfonation of dopamine, which is often used as its probe substrate. A new method for analyzing dopamine-3-O-sulfate and dopamine-4-O-sulfate by high-performance liquid chromatography was developed and the enzyme kinetic parameters for their formation were determined using purified recombinant human SULT1A3. The results show that SULT1A3 strongly favors the 3-hydroxy group of dopamine, which indicates that it may be the major enzyme responsible for the difference between the circulating levels of dopamine sulfates in human blood. All 19 known human UGTs were expressed as recombinant enzymes in baculovirus infected insect cells and their activities toward dopamine and estradiol were studied. UGT1A10 was identified as the only UGT capable of dopamine glucuronidation at a substantial level. The results were supported by studies with human intestinal and liver microsomes. The affinity was low indicating that UGT1A10 is not an important enzyme in dopamine metabolism in vivo. Despite the low affinity, dopamine is a potential new probe substrate for UGT1A10 due to its selectivity. Dopamine was used to study the importance of phenylalanines 90 and 93 in UGT1A10. The results revealed distinct effects that are dependent on differences in the size of the side chain and on the differences in their position within the protein. Examination of twelve mutants revealed lower activity in all of them. However, the enzyme kinetic studies of four mutants showed that their affinities were similar to that of UGT1A10 suggesting that F90 and F93 are not directly involved in dopamine binding in the active site. The glucuronidation of β-estradiol and epiestradiol (α-estradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. The results show that there are clear differences in the regio- and stereoselectivities of UGTs. The most active isoforms were UGT1A10 and UGT2B7 demonstrating opposite regioselectivity. The stereoselectivities of UGT2Bs were more complex than those of UGT1As. The amino acid sequences of the human UGTs 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. Several mutants were constructed to identify the residues responsible for the activity differences. The results revealed that the residues between Leu86 and Tyr176 of UGT1A9 determine the differences between UGT1A9 and UGT1A10. Phe117 of UGT1A9 participated in 1-naphthol binding and the residues at positions 152 and 169 contributed to the higher glucuronidation rates of UGT1A10. In summary, the results emphasize that the substrate selectivities, including regio- and stereoselectivities, of UGTs are complex and they are controlled by many amino acids rather than one critical residue.