a) Soluble adenylate cyclase (30 rg) depleted of Lubrol PX by sucrose density centrifugation as described in the legend to above graph was assayed in 200 ~1 with 0.1 mM Gpp(NH)p and in the presence of varying amounts (0 to 300 pg) of Lubrol PX. The ratio (w/w) of Lubrol: protein is given on the abscissa. Separation of solubilized membrane proteins by sucrose density gradient centrifugation. Membranes (8.8 mg of protein) were incubated in 1.5 ml of Buffer A with 1 rnM GMP and 50 pM DLisoproterenol for 30 min at 37”. After three washes with 4 ml of Buffer B, the membranes were solubilized with 20 mM Lubrol PX as described in Ref. 6. The solubilized material was concentrated to a volume of 200 ~1 by centrifugation at 1000 x g in a “centrifilo Filter cone” device (CF 25-Amicon) and layered on top of 5 ml of a 5 to 30% linear sucrose gradient in Buffer B and centrifuged in a SW 50 rotor (Beckman) at 48,000 rpm for 14 h at 4”. Fractions of 0.25 ml were collected and adenylate cyclase activity was measured in 200 ~1 in the presence of 0.1 mM Gpp(NH)p and 0.5 mM Lubrol PX as described under “Methods.” Binding of 13HlGpp(NH)p was determined in lOO- ~1 aliquots according to Pfeuffer and Helmreich (6) and protein was measured as described under “Methods.”
b) Transient transfection of COS-1 or HEK 293 cells with increasing amounts of either wild type αs or N54-αs cDNA resulted in comparable increases in αs immunoreactivity with little or no change in Gβ subunit level. This increased expression of αs protein was associated with increased basal cAMP accumulation, and the mutant was slightly more effective than wild type αs. In contrast, cellular cAMP levels were less affected by isoproterenol in the presence of the mutant than wild type αs. Consequently, agonist-stimulated cAMP levels were slightly but consistently less in both cell lines transfected with the mutant than in cells transfected with wild type αs.
c) In conclusion, heterotrimeric GTP binding proteins, or G proteins, transduce signals from receptors on the surface of the cell to various intracellular effector enzymes. The superfamily of GTP binding proteins, which includes among others the α subunits of heterotrimeric G proteins and the monomeric GTP binding proteins, share four conserved amino acid sequences that form the core of the GTP regulatory mechanism of these proteins [1, 2, 3]. The consensus sequence of the first conserved region of the guanine nucleotide binding site is GX4GK(S/T), which includes Ser-17 in Ras and the equivalent Ser-54 of Gαs
