Study of GDNF-Family receptor alpha 2 and inhibitory activity of GDNF-family receptor alpha 2b (GFR alpha 2b) isoform

Abstract

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. GDNF and NTN exert their effects through a multi-component receptor system consisting of the GDNF family receptor alpha (GFRN1) and the co-receptor RET and/or NCAM. GDNF preferentially binds to GFRN11, while GFRN12 is the cognate receptor for NTN. This study focused on the biochemical and morphological effects of ligand-activated GFRN11 and GFRN12 isoforms. In the initial part of the study, GDNF and NTN were found to activate distinct miRNA precursors in cells endogenously expressing RET, NCAM and GFRN12 but not GFRN11, indicative of specificity in ligand-receptor cross-talk. There are at least three alternatively spliced isoforms of GFRN12 in the nervous system: GFRN12a, GFRN12b, and GFRN12c. Quantitation using highly specific and sensitive quantitative real-time PCR revealed comparable expression levels of these isoforms in various regions of the human brain, lending evidence to the idea that the isoforms may have physiological roles in the nervous system. These isoforms showed ligand-selectivity in MAPK (ERK1/2) and Akt signaling, and regulated different early response genes. When stimulated with GDNF or NTN, both GFRN12a and GFRN12c, but not GFRN12b, promoted neurite outgrowth in transfected Neuro2A cells. In co-expression studies, GFRN12b was found to inhibit ligand-induced neurite outgrowths mediated by GFRN12a, GFRN12c, and GFRN11a, another member of the GDNF family receptor. Furthermore, activation of GFRN12b also inhibited neurite outgrowths induced by retinoic acid and the inhibitory activities were RhoA dependent. On the other hand, the ligand-induced neurite outgrowths through GFRN12a and GFRN12c isoforms showed distinct signaling mechanisms. Differential biochemical and neuritogenic activities also exist with the GFRN11 receptor isoforms, GFRN11a and GFRN11b. When co-expressed, GFRN11b antagonized neurite outgrowth mediated by GFRN11a, in a RhoA-ROCK dependent manner. The results from this study suggest a novel paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. Thus, depending on the expressions of specific GFRN12 and GFRN11 receptor spliced isoforms, GDNF and NTN may promote or inhibit neurite outgrowth through the same multi-component receptor complex. The emerging view is that the combinatorial interactions of the spliced isoforms of GFRN11, GFRN12, RET and NCAM may contribute to the complexity of multi-component signaling system and produce a myriad of observed biological responses.