MOLECULAR CLONING OF A NOVEL MURINE ISOFORM OF THE GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR RECEPTOR

Abstract

Neurotrophic factors are crucial for the development and maintenance of the nervous system. Recently, one more important and potential neurotrophic factor has been added to the family with the discovery of glial cell line-derived neurotrophic factor (GDNF). GDNF was initially isolated and purified from B49, a rat glial cell line and was shown to have trophic effects on cultured midbrain dopaminergic neurons of substantia nigra. The dopaminergic neurons in midbrain of Parkinson's patients degenerate and therefore, there was a hope that GDNF could be a potential therapeutic agent for the treatment of Parkinson's disease. It is now known that GDNF is a potent factor for the survival of a number of central and peripheral neurons including the development of kidney and enteric nervous system. Critical preclinical evaluations of GDNF have been conducted in various animal models of Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Alzheimer's disease (AD). The hope is that GDNF could be used for the treatment of these diseases in the near future. Before developing GDNF as a drug it is very important and necessary to understand the basic mode of action of this factor. To understand the mechanism of action of GDNF, the mouse GDNF receptor was cloned and characterized in the present study. Multiple tissue Northern blot analysis of various mouse tissues, including the brain, demonstrated the existence of multiple transcripts of GDNF receptor. Screening of an adult mouse liver cDNA library yielded two isoforms of the receptor. One of the forms GDNFR? showed a high degree of homology with other mammalian GDNFR?, and the other was a novel form herein named as GDNFR?. GDNFRP is identical to that of the GDNFR? except for a deletion of five amino acids. These two forms were found to share high homologies with the recently isolated GDNF family receptors GFR?-2, GFR?-3, and GFR?-4. Tissue specific expression studies using RT-PCR demonstrated the presence of both the GDNFR? and GDNFR? in the various murine tissues except muscle. Two different fragments of the coding region of the mouse GDNFR? was subcloned into pQE-8 and expressed in E. coli. The expressed proteins were found exclusively in the inclusion body of the E. coli. These proteins were purified and used to raise specific antiserum. The specificity of these antisera was tested by western blots.