Mutations in .trimcric G proteins can cause many diseases in humans. Patients with acromegaly often have pituitary

Question:

Mutations in .trimcric G proteins can cause many diseases in humans. Patients with acromegaly often have pituitary tumors that oversecrete growth hormone (GH). GH-releasing hormone (GHRH) stimulates GH release from the pituitary by binding to GHRH receptors and stimulating adenylyl cyclase. Researchers wanted to know whether mutations in G_as, played a roll in this condition. Cloning and sequencing of the wild-type and mutant G_as'gene from normal individuals and patients with the pituitary tumors revealed a missense mutation in the G_asgene sequence.

a. To investigate the effect of the mutation on G_as, activity, wild-type and mutant G_as, cDNAs were transfected into cells that lack the G_as, gene. These cells express a Î²-radrenergic receptor, which can be activated by isoproterenol, a Î²₂- adrenergic receptor agonist. Membranes were isolated from transfected cells and assayed for adcnylyl cyclase activity in the presence of GTP or the hydrolysis-resistant GTP analog, GTP€”Î³S. from the figure below, what do you conclude about the effect of the mutation on G_as, activity in the presence of GTP alone compared with GTP€”Î³S alone or GTP plus isoproterenol ( iso)?

250 200 150 100 GTP GTP+iso GTPS GTP GTP+iso GTPYS Wild type Mutant Adenylyl cyclase activity

b. In the transfected cells described in part (a), what would you predict would be the cAMP levels in cells transfccted with the wild-type G_as , and the mutant G_as,? What effect might this have on the cells?

c. To further characterize the molecular defect caused by this mutation, the intrinsic GTP_ase activity present in both wild-type and mutant G_as, was assayed. Assays for GTP_ase activity showed that the mutation reduced the k_cat-GTP (catalysis rate constant for GTP hydrolysis) from a wild-type value of 4.1 min^-1 to the mutant value of 0.1 min^-1. What do you conclude about the effect of the mutation on the GTP_ase activity present in the mutant G0 , subunit? How do these GTP_ase results explain the adenylyl cyclase results shown in part (a)?