G protein-coupled receptor (GPCR) regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by mitogen-activated protein kinases (MAPK). This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signalling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms we have elucidated how pituitary gonadotrope cells decode an input gonadotropin releasing hormone (GnRH) signal into coherent transcriptional output from the luteinizing hormone (LH) -subunit gene promoter. We show that GnRH activates c-Src, and multiple members of the MAPK family, c-Jun NH2-terminal kinase 1/2 (JNK), p38MAPK and ERK1/2. Using dominant negative point mutations and chemical inhibitors we identified calcium dependent proline-rich tyrosine kinase 2 (Pyk2) specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2 and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH -subunit gene proximal promoter (PP), which was also activated by p38MAPK, but not JNK. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity and DNA binding. We have identified a novel GnRH activated Pyk2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH -subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression.