Three intragenic suppressors of a GTPase-deficient allele of GNAS associated with McCune–Albright syndrome

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   Figure 1

   Basal cAMP levels in cells transfected with the suppressors or Arg→His mutations originally identified in a yeast screen. (A) Immunoblot of lysates from cells transfected with the indicated alleles of Gs probed for Gs and GAPDH. (B) Basal cAMP levels from cells transiently transfected with 5 μg of plasmid encoding the indicated alleles of Gs. Bars represent the mean±s.e.m. for three to four independent experiments, each performed in duplicate. *Significantly lower than R201H basal cAMP, P<0.05 and **not significantly different from WT basal cAMP, P>0.05.
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   Figure 2

   Basal and hormone-stimulated cAMP production in cells expressing intragenic suppressors of R201H. (A) Immunoblot of lysates from cells transfected with the indicated alleles of Gs probed for Gs and GAPDH. (B) Basal and maximally-stimulated cAMP levels from (C). Data are the mean±s.e.m. for three independent experiments, each performed in duplicate. *Statistically lower than WT hCG-stimulated levels, P<0.05. (C) Dose–response curves for cAMP production in cells cotransfected with the human luteinizing hormone receptor (LHR) and the indicated alleles of Gs. Filled circles, vector; open squares, WT Gs; open circles, R201H/R231C; filled diamond, R201H/F142S; and open triangle, R201H/L266T. Points represent the mean±s.e.m. for three independent experiments, each performed in duplicate.
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   Figure 3

   All mutations at F142 suppress the constitutive activity of R201H. (A) Immunoblot of lysates from cells transfected with the various alleles of Gs probed for Gs and GAPDH. (B) Basal cAMP levels from cells transiently transfected with 5 μg of plasmid encoding the indicated alleles of Gs. Bars represent the mean±s.e.m. for three to four independent experiments, each performed in duplicate. *Not different from WT basal cAMP, P>0.05.
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   Figure 4

   Several mutations at R231 suppress the constitutive activity of R201H. (A) Immunoblot of lysates from cells transfected with the various alleles of Gs probed for Gs and GAPDH. (B) Basal cAMP levels from cells transiently transfected with 5 μg of plasmid encoding the indicated alleles of Gs. Bars represent the mean±s.e.m. for four independent experiments, each carried out in duplicate. *Not different from WT basal cAMP, P>0.05.
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   Figure 5

   Several mutations at L266 suppress the constitutive activity of R201H. (A) Immunoblot of lysates from cells transfected with the various alleles of Gs probed for Gs and GAPDH. (B) Basal cAMP levels from cells transiently transfected with 5 μg of plasmid encoding the indicated alleles of Gs. Bars represent the mean±s.e.m. for three independent experiments, each carried out in duplicate. *Not different from WT basal cAMP, P>0.05.
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   Figure 6

   Mutation in the suppressor sites alone does not alter the ability of cells to respond to hormone. (A) Immunoblot of lysates from cells transfected with the indicated alleles of Gs probed for Gs and GAPDH. (B) Basal and maximally-stimulated cAMP levels from (A) above. Data are the mean±s.e.m. for three independent experiments, each carried out in duplicate. The single mutants were not statistically different from WT in basal cAMP levels or cAMP levels produced in response to 10 U/ml hCG. (C) Dose–response curves for cAMP production in cells cotransfected with the human luteinizing hormone receptor (LHR) and the indicated alleles of Gs. Filled circles, vector; open circles, WT Gs; filled triangle, R201H; open down-pointing triangle, R231C; open diamond, F142S; and filled square, L266N. Points represent the mean±s.e.m. for three independent experiments, each performed in duplicate.
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   Figure 7

   The locations of the three suppressor mutations are modeled on the GTPγS-bound structure of Gs (AZT1) using Chimera Software (University of California, San Francisco, CA, USA). (A, B and C) Structures of the entire Gs protein. Switch 1 is yellow, Switch II is pink, and Switch III is green. The nucleotide is shown as ball-and-stick; R201H and the indicated suppressor sites are shown in space-fill. The structures are rotated slightly left to right in (A, B and C) to provide better visualization of the location of each suppressor mutation. (D, E and F) Closeups of the local region of each suppressor mutation. (D) Interaction of Arg231 with other residues in the Ras-like domain. Hydrogen bonds are indicated in blue. Arg231 is shown colored by atom, extending from left to right from the Switch II helix. Arg231 forms a salt bridge with Glu268 (also shown colored by atom). Arg231 also hydrogen bonds to the backbone of Switch II and a water molecule. (E) Interaction of Leu266 with other amino acids in the Ras-like domain. Leu266 is shown in red, Leu270 in blue, and Phe312 is yellow (not all atoms were mapped on the structure for Phe312). These three residues pack with hydrophobic interactions in the core of the Ras-like domain of Gs. (F) Interactions of Phe142 with other residues in the helical domain. Phe142 is colored blue; Phe140 is colored yellow, and Phe146 is colored orange form a hydrophobic triad with Phe142. (G) Alignment of the helical domains of GTPγS-bound Gs (1AZT) and nucleotide-free Gs (3SN6). The blue chain represents the GTPγS-bound structure, with cyan residues showing the cluster of three phenylalanine residue side chains in the helical domain. The nucleotide-free structure is colored tan, with phenylalanine side chains in the helical domain colored yellow. The helical domain is to the left on the figure, showing little change in the conformations of the two protein states. The Ras-like domain is on the right of the figure, showing the large rotation of this domain relative to the helical domain in the two structures.

• © 2014 Society for Endocrinology