Molecular and functional characterization of novel CRFR1 isoforms from the skin.

In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1alpha, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1alpha was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1alpha-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis-elements (CRE, CaRE, SRE, AP1 or NF-kappaB) demonstrated that only CRFR1alpha was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4beta-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction.