Glu2.53(90) of the GnRH receptor is part of the conserved G protein-coupled receptor structure and does not form a salt-bridge with Lys3.32(121).

GnRH receptor mutations, Glu2.53(90)Lys and Glu2.53(90)Asp, cause congenital hypogonadotropic hypogonadism. The Glu2.53(90) side-chain has been proposed to form an intramolecular salt-bridge with Lys3.32(121), but conserved intramolecular interaction networks in G protein-coupled receptor crystal structures predict that it interacts with Ser3.35(124) and Trp6.48(280). We investigated interhelical interactions of Glu2.53(90) that stabilise GnRH receptor folding using functional analyses and computational modelling of mutant receptors. The Glu2.53(90)Asp mutant was non-functional, but mutants with hydrophobic amino acids or Arg substituted for Glu2.53(90) were functional, excluding a salt-bridge interaction. The Glu2.53(90)Arg and Trp6.48(280)Arg mutants had decreased affinity for GnRH. Models showed that congenital Glu2.53(90)Lys and Glu2.53(90)Asp mutations disrupt interactions with Ser3.35(124) and Trp6.48(280) respectively, whereas the Glu2.53(90)Arg and Trp6.48(280)Arg mutations preserve intramolecular contacts, but increase distance between the transmembrane helices. Our results show that disruption of interhelical contacts that are conserved in G protein-coupled receptors accounts for the effects of some disease-associated GnRH receptor mutations.

Tumor cell culture survival following glucose and glutamine deprivation at typical physiological concentrations.

OBJECTIVE: Most glucose (and glutamine)-deprivation studies of cancer cell cultures focus on total depletion, and are conducted over at least 24 h. It is difficult to extrapolate findings from such experiments to practical anti-glycolytic treatments, such as with insulin-inhibiting diets (with 10%-50% carbohydrate dietary restriction) or with isolated limb perfusion therapy (which usually lasts about 90 min). The aim of this study was to obtain experimental data on the effect of partial deprivation of d-glucose and l-glutamine (to typical physiological concentrations) during 0 to 6-h exposures of HeLa cells. METHODS: HeLa cells were treated for 0 to 6 h with 6 mM d-glucose and 1 mM l-glutamine (normal in vivo conditions), 3 mM d-glucose and 0.5 mM l-glutamine (severe hypoglycemic conditions), and 0 mM d-glucose and 0 mM l-glutamine ("starvation"). Polarization-optical differential interference contrast and phase-contrast light microscopy were employed to investigate morphologic changes. RESULTS: Reduction of glucose levels from 6 to 3 mM (and glutamine levels from 1 to 0.5 mM) brings about cancer cell survival of 73% after 2-h exposure and 63% after 4-h exposure. Reducing glucose levels from 6 to 0 mM (and glutamine levels from 1 to 0 mM) for 4 h resulted in 53% cell survival. CONCLUSION: These data reveal that glucose (and glutamine) deprivation to typical physiological concentrations result in significant cancer cell killing after as little as 2 h. This supports the possibility of combining anti-glycolytic treatment, such as a carbohydrate-restricted diet, with chemotherapeutics for enhanced cancer cell killing.