Roles of Pyk2 in signal transduction after gonadotropin-releasing hormone receptor stimulation.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic neurons. It has been reported that GnRH treatment of cultured GnRH neurons (GT1-7 cells) activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in the activation of extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 (ERK1/2). In the present study, we first examined the possibility that GnRH treatment might activate epidermal growth factor receptor (EGFR). We found that activation of EGFR after GnRH treatment for 5 min was much less than after EGF or heparin-binding EGF treatment. Next, we examined whether or not Pyk2 bound to growth factor receptor-binding protein 2 (Grb2). We overexpressed FLAG-fused Pyk2 in GT1-7 cells, and immunoprecipitated Pyk2 using an anti-FLAG antibody. The binding of Pyk2 to Grb2 was detected only after GnRH treatment. In contrast, a site-directed mutant of Pyk2 wherein tyrosine 881 was mutated to phenylalanine did not bind to Grb2. Studies with small interfering RNA and inhibitors indicated that the activation of Grb2/Ras/Raf/MEK was a major pathway to ERK1/2 activation after the short-term treatment of GT1-7 cells with GnRH.