Receptor tyrosine kinases (RTKs) activate the mitogen-activated protein (MAP) kinases called extracellular signal-regulated kinases 1 and 2 (ERK1/2) through a signaling cascade involving proteins such as Shc, Grb2, SOS, Ras, Raf-1 and MEK. Activation of ERK1/2 directs proliferation and differentiation in a wide array of cell types. G-protein-coupled receptors (GPCRs) of many types have been shown to activate the ERK cascade, but the complete mechanisms are yet to be elucidated. Gs-coupled receptors activate adenylyl cyclase (AC) leading to a rapid increase in cyclic AMP (cAMP), and the main targets for cAMP are protein kinase A (PKA) and the Exchange Protein directly Activated by cAMP (Epac) specific for the small G-protein Rap1. It has been proposed for the Gs-coupled â2-adrenergic receptor that activation of ERK1/2 proceeds through Rap1 and B-Raf in a manner independent of Ras. However, for the human Gs-coupled serotonin receptors 5-HT4(b) and 5-HT7(a) it has been shown that ERK1/2 activation occurs independently of Rap1, relying instead on PKA and Ras. In this project we have explored various signaling mechanisms originating from the human Gs-coupled 5-HT7 serotonin receptors, with particular interest in the mechanisms of activation of ERK1/2. We demonstrate the activation of ERK1/2 in HEK293 cells transfected with 5-HT7(b) and 5-HT7(d) receptors subsequent to stimulation with serotonin. For the 5-HT7(b) receptors, we show that the observed activation of ERK1/2 is dependent on cAMP and Ras, but not on Epac and Rap1. Furthermore, direct cytosolic calcium measurements have shown that treatment with serotonin leads to a rapid but transient elevation in intracellular calcium in HEK293 cells stably transfected with 5-HT7(b) receptors, and the observed phosphorylation of ERK1/2 is mediated, at least partly, through a calcium-dependent pathway. We show that HEK293 cells endogenously express the Ras specific, Ca2+/calmodulin-dependent guanine nucleotide exchange factor Ras-GRF1. Ras-GRF1 becomes phosphorylatedsubsequent to 5-HT stimulation of 5-HT7(b) receptors, and it has previously been demonstrated that this phosphorylation enhances its towards Ras. This indicates that Ras-GRF1 has a role in the observed Ras-dependent activation of ERK1/2 mediated through 5-HT7(b) receptors.