G Protein-Coupled Signal Transduction

Question 1

hormone stimulated adenylyl cyclase

Answer 1

-you could convert ATP into cyclized molecule of cAMP- 3’ hydroxyl group attacks alpha phosphate of ATP- you get rid of this pyrophosphate and make cyclized molecule
-molecule made in response to range of diff hormones like glucagon and epinephrine

Question 2

activation of adenylyl cyclase

Answer 2

not only could you stimulate this adenylyl cyclase activity with hormones but you could also stimulate with NaFl

Question 3

guanine nucleotides in cAMP production

Answer 3

Ex. if you look at the binding of radio labelled glucagon
-introduce guanine nucleotides you could change the affinity of membranes to glucagon
-guanine nucleotides could enhance glucagon-stimulated adenylase activity and they stimulated adenylyl cyclase on their own but didn’t do anything to NaFl
-glucagon plus GTP you get maximal activity
-guanine nucleotides’ ability to be hydrolyzed seemed to be part of this because non-hydrolyzable analogs could activate adenylyl cyclase irreversibly
-hydrolyzable version of GTP could competitively antagonize the irreversible GTP analog

Question 4

GTPase and AC activation

Answer 4

-GTP in the setting where hormone was stimulated in adenylyl cyclase activity you could trigger hydrolysis of GTP
-ADP ribosylation factor- cholera toxin has 2 effects in this system: reduced GTP hydrolysis induced by isoproterenol but it could also enhance AC activation
-some kind of cycle with active form of adenylyl cyclase enzyme associated with GTP and inactive form of GDP –> hydrolyze GTP you go down to GDP but if you don’t have hydrolyzed GTP go up to active form
-cholera toxin was locking in the active form

Question 5

discovery of G protein

Answer 5

cell line deficient in fluoride-stimulated AC
-added protein complex to cyclase deficient membranes you could reconstitute ability of fluoride or other hormone to stimulate adenylyl cyclase
-protein = heterotrimer with 3 subunits –> relatively larger alpha subunits with site that binds GTP and beta and gamma subunits that stick together
-all 3 can be isolated as heterotrimer but if you force them apart, alpha subunit comes apart and beta and gamma subunits stick together
-alpha subunit binds hydrolyzable GTP with enzymatic activity- hydrolyze GTP to GDP
-cholera toxin- transfers nicotinamide adenine dinucleotide group onto alpha subunit and prevents it from hydrolyzing GTP