PMID- 16696853 OWN - NLM STAT- MEDLINE DCOM- 20060721 LR - 20231213 IS - 0022-3042 (Print) IS - 1471-4159 (Electronic) IS - 0022-3042 (Linking) VI - 97 IP - 5 DP - 2006 Jun TI - A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions. PG - 1447-55 AB - The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4. FAU - Swick, Lance AU - Swick L AD - Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA. FAU - Kapatos, Gregory AU - Kapatos G LA - eng GR - R01 NS026081-18/NS/NINDS NIH HHS/United States GR - NS 26081/NS/NINDS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PL - England TA - J Neurochem JT - Journal of neurochemistry JID - 2985190R RN - 0 (AHA1 protein, S cerevisiae) RN - 0 (GCHFR protein, human) RN - 0 (HSP90 Heat-Shock Proteins) RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (Polymers) RN - 0 (Saccharomyces cerevisiae Proteins) RN - 0 (Biopterins) RN - 31C4KY9ESH (Nitric Oxide) RN - 86-01-1 (Guanosine Triphosphate) RN - EC 1.14.13.39 (Nitric Oxide Synthase Type III) RN - EC 3.5.4.16 (GTP Cyclohydrolase) RN - EC 3.6.1.- (Chaperonins) RN - EGX657432I (sapropterin) SB - IM MH - Amino Acid Sequence/physiology MH - Binding Sites/physiology MH - Biopterins/analogs & derivatives/biosynthesis MH - Chaperonins MH - Crystallography, X-Ray MH - Endothelium, Vascular/enzymology MH - Enzyme Activation/physiology MH - GTP Cyclohydrolase/*chemistry/genetics/*metabolism MH - Gene Library MH - Guanosine Triphosphate/*metabolism MH - HSP90 Heat-Shock Proteins/metabolism MH - Humans MH - Intracellular Signaling Peptides and Proteins/*metabolism MH - Molecular Sequence Data MH - Nitric Oxide/biosynthesis MH - Nitric Oxide Synthase Type III/metabolism MH - Polymers/metabolism MH - Protein Binding/physiology MH - Protein Structure, Secondary/physiology MH - Protein Structure, Tertiary/physiology MH - Saccharomyces cerevisiae/metabolism MH - Saccharomyces cerevisiae Proteins/metabolism MH - Two-Hybrid System Techniques PMC - PMC2239266 MID - NIHMS37432 EDAT- 2006/05/16 09:00 MHDA- 2006/07/22 09:00 PMCR- 2008/02/12 CRDT- 2006/05/16 09:00 PHST- 2006/05/16 09:00 [pubmed] PHST- 2006/07/22 09:00 [medline] PHST- 2006/05/16 09:00 [entrez] PHST- 2008/02/12 00:00 [pmc-release] AID - JNC3836 [pii] AID - 10.1111/j.1471-4159.2006.03836.x [doi] PST - ppublish SO - J Neurochem. 2006 Jun;97(5):1447-55. doi: 10.1111/j.1471-4159.2006.03836.x.