GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme of a metabolic pathway synthesizing tetrahydrobiopterin (BH(4)), the cofactor dimerizing and activating inducible nitric oxide synthase (NOS-2). GCH1 protein expression and enzyme activity are minimal in cultured, phenotypically stable, untreated normal adult human astrocytes (NAHA), but are strongly induced, together with NOS-2, by a mixture of three proinflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma--the CM-trio) released by microglia under brain-damaging conditions. The resulting hyper-production of NO severely harms neurons. In this study, using MALDI-TOF/MS, PMF, Western immunoblotting (WB), and antibody microarrays we identified several proteins coimmunoprecipitating with GCH1. Under basal conditions, GCH1 was associated with various adaptor/regulator molecules involved in G-protein-coupled receptors signalling, protein serine/threonine phosphatase 2Cbeta (PP2Cbeta), and serine-threonine kinases like Ca(2+) calmodulin kinases (CaMKs), casein kinases (CKs), cAMP-dependent kinases (PKAs), and mitogen-activated protein kinases (MAPKs). Exposure to the three cytokines' mixture (CM-trio) significantly changed, within the 48-72 h required for the induction and activation of GCH1, the levels and identities of some of the 0 h-associated proteins: after 72 h CK-IIalpha tended to dissociate from, whereas MAPK12 and JNK3 were strongly associated with fully active GCH1. These findings provide a first enticing glimpse into the intricate mechanisms regulating GCH1 activation by proinflammatory cytokines in NAHA, and may have therapeutic implications.