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. Activated Pyk2 activates ERK1/2 through the Grb2/Ras/Raf/MEK pathway. GnRH long term treatment induces the shedding of proHB‐EGF.