Physical evidence of the coupling of solubilized 5-HT1A binding sites with G regulatory proteins.
Previous investigations (El Mestikawy et al., J Neurochem 51: 1031-1040, 1988) have shown that 5-HT1A binding sites (R[5-HT1A]) solubilized by CHAPS from rat hippocampal membranes can be modulated by guanine nucleotides, as expected from their solubilization together with associated G regulatory proteins (G). Studies of the hydrodynamic properties of solubilized R[5-HT1A] have been presently carried out in order to assess in a more direct way the presence of R[5-HT1A]-G complexes in the soluble extract. Under control conditions, the sedimentation of a CHAPS extract from hippocampal membranes through a 5-30% sucrose gradient (200,000 g, 17 hr, 4 degrees) gave two maxima of [3H]8-OH-DPAT binding activity corresponding to sedimentation coefficients of 8.0 S and 10.0 S, respectively. Running the gradient in the presence of 1 microM GTP revealed a significant reduction of the 10.0 S peak, as expected from the loss of material (probably a G protein) normally associated with R[5-HT1A]. Conversely, attempts to prevent the dissociation of R[5-HT1A]-G by treatment of CHAPS soluble hippocampal extracts with the cross-linking reagent disuccinimidyl suberate (0.1 mM) resulted in a significant increase (+70%) in [3H]8-OH-DPAT binding activity associated with the appearance of a new sedimenting material with a higher coefficient (16.5 S). Furthermore, [3H]8-OH-DPAT binding became almost completely insensitive to guanine nucleotides as expected from the irreversible coupling by disuccinimidyl suberate of R[5-HT1A] with G protein(s). WGA-agarose chromatography of CHAPS soluble hippocampal extract supplemented with GTP allowed the physical separation of R[5-HT1A] from the bulk of G proteins, and a concomitant decrease of [3H]8-OH-DPAT high affinity binding capacity. Partial recovery of the latter could be achieved by reconstituting R[5-HT1A]-G complexes upon the addition of a mixture of pure bovine Gi + Go to G-deprived soluble extracts. Finally in vivo treatment with Pertussis toxin (5 micrograms intracerebroventricularly, 48 hr before killing) resulted in a significant reduction of the specific binding of [3H]8-OH-DPAT (-36%) to hippocampal membranes and corresponding CHAPS soluble extracts, and a marked decrease in the inhibitory effect of GppNHp. Accordingly the G protein associated with R[5-HT1A] belongs probably to the Gi or Go families.
Emerit
M B
MB
INSERM U.288, Neurobiologie Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpétrière, Paris, France.
el Mestikawy
S
S
Gozlan
H
H
Rouot
B
B
Hamon
M
M
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000818:Animals; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D003902:Detergents; D019204:GTP-Binding Proteins / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006624:Hippocampus / Q000032:analysis* / Q000648:ultrastructure; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013388:Succinimides / Q000494:pharmacology; D013764:Tetrahydronaphthalenes / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D011985:Receptors, Serotonin; D013388:Succinimides; D013764:Tetrahydronaphthalenes; D006165:Guanylyl Imidodiphosphate; C022581:Bolton-Hunter reagent; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C019358:disuccinimidyl suberate