1. (8 points) In studies trying to understand the regulation of hormone-sensitiv

Question

1. (8 points) In studies trying to understand the regulation of hormone-sensitive adenylate cyclase, it was discovered that non-hydrolyzable GTP analogs and cholera toxin (that inhibits the GTPase activity of Gs) were able to activate the enzyme in a sustained (irreversible) profile, therefore indicating that GTP hydrolysis represents a deactivation (turn-off) regulatory step.

a) Will these analogs or toxin have any effect on adenylate cyclase activity in the absence of any added hormone?

b) If you add an factor that increases the rate of GTPase hydrolysis of the Gs protein, will this activate or inhibit the observed downstream effect upon addition of the hormone (this is in the absence of GTP analogs or toxin). Explain your answer.

Explanation / Answer

a) C6 glioma cells and B104 neuroblastoma cells both possess adenylate cyclase activity, but only C6 cells have beta-adrenergic receptors. However, when cocultured with B104 cells, C6 cells show a marked decrease in their ability to accumulate cyclic AMP upon stimulation with beta-agonists. Since both beta-receptors and cholera toxin-stimulated adenylate cyclase activities are present in C6 cells of ''C6/B104'' cocultures, we conclude that the beta-receptor/adenylate cyclase transduction mechanism in cocultured C6 cells is uncoupled.

C6 glioma cells and B104 neuroblastoma cells both possess adenylate cyclase activity, but only C6 cells have ..beta..-adrenergic s. However, when cocultured with B104 cells, C6 cells show a marked decrease in their ability to accumulate adenosine 3',5'-monophosphate upon stimulation with ..beta.. receptor agonists. Since both ..beta.. receptors and cholera toxin-stimulated adenylatecyclase activities are present in C6/B104 cocultures, we conclude that the ..beta.. receptor/adenylate cylase transduction mechanism in cocultured C6 cells in uncoupled.Summary Coupling of the previously characterized b1- and b2-like adrenoceptors in the sea pansy Renilla koellikeri with adenylatecyclase was examined in membrane preparations from this cnidarian. Adenylate cyclase activity was stimulated by several guanine nucleotides, such as GTP, Gpp(NH)p and GTPgS. Fluoride ions and cholera toxin greatly enhanced the enzyme activity, whereas forskolin had no effect on basal.

b)

Activated GPCRs cause a conformational change in the attached G protein complex, which results in the Gs alpha subunit's exchanging GDP for GTP and separation from the beta and gamma subunits. The Gs alpha subunit, in turn, activates adenylyl cyclase, which quickly converts ATP into cAMP. This leads to the activation of the cAMP-dependent pathway. This pathway can also be activated downstream by directly activating adenylyl cyclase or PKA.

The Gs alpha subunit slowly catalyzes the hydrolysis of GTP to GDP, which in turn deactivates the Gs protein, shutting off the cAMP pathway. The pathway may also be deactivated downstream by directly inhibiting adenylyl cyclase or dephosphorylating the proteins phosphorylated by PKA.

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