|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
We identified an activating mutation, Asp-578*Gly of the lutropin receptor (LHR) in familial male precocious puberty (FMPP) for the first time. This is a mutation causing constitutive activation (CA) without stimulation by hormones. We substituted other amino acids for Asp-578. Mutation to Asn did not, but mutation to Glu showed CA, proving that the hydrogen bonding formed by beta-C=O group of Asp-578 contributes to maintaining the inactive conformation of the LHR.By computer modeling, we identified a possible partner of the hydrogen bonding, Asn-619. When Asn-619 was mutated to Glu or Gln, CA occurred. D578S (Asp-578*Ser) showed CA, but a double mutation, D578S+N619Q (Asp-578*Ser + Asn-619 *Gln) did not show CA, but showed a characteristics similar to the wild-type LHR.The summation of the two side chains of the 578th and 619th amino acids is the same as that of Asp-578 and Asn-619 in the wild-type LHR.Ser and Gin can form a hydrogen bonding like Asp and Asn. Three dimensional distribution of Asp-578 and Asn-619 is the same as expected by the computer modeling and the existence of a hydrogen bonding between the two residues was confirmed.
LHR mutations involving Asp-564 were also identified in FMPP.Asp^<564>* Glu did not, but Asp^<564>* Asn showed CA, suggesting the importance of negative charge in this residue for maintaining the inactive conformation of the LHR.The degree of CA was additive in double mutants with Asp^<578>, suggesting an interhelical bond network other than that formed by Asp^<578>.
We confirmed similar hydrogen bondings in the thyrotropin receptor that is closely related G-protein-coupled receptor to the LHR.