Endocrinology Second Messengers: CAMP IP3 DAG Recap: 1) Ligand binds to GPCR 2) If ligand is an agonist, it activates the GPCR 3) GPCR is able to talk to intracellular G-proteins 4) G-protein becomes activated- this can be detected because a-subunit has GTP bound. Also a-subunit dissociates with By-subunit. 5) a-subunit is a GTPase and therefore kicks off a phosphate to produce GDP. Therefore, reassociation of ??? 27th October 2016 Ligand Activated receptor H2N Extracellular face Cytosolic face COOH Stimulatory G protein Classically, G-Proteins are Divided into 4 Families Gi - Adenylate cyclase Gs - Adenylate cyclase Gq - phospholipase C G12/13 Go - ion channels The division is based on the similarity of there alpha subunits. The Adenylate Cyclase - CAMP System Adenylate Cyclase - enzyme that makes cAMP BY GTP GDP GTP-GDP exchange Membrane protein with a G-protein activator - It is activated by Gs and inactivated by Gi (and Go) Adenylate Cyclase is a membrane protein, but works as an enzyme, as it converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). CAMP is a second messenger. In mammals, there are 10 different types of Adenylate Cyclases known. Agonist Regulation of Adenylate Cyclase Physiological regulation: Gas is stimulatory and is able to activate Adenylate cyclase Got Gai is inhibitory and is able to inhibit Adenylate cyclase activation. It binds directly to Adenylate cyclase. Adenylate Cyclase ATP CAMP The beta-gamma complex modulates the activity of Adenylate cyclase.
Endocrinology 27th October 2016 Calcium can also modulate Adenylate cyclase - it depends on the form. Experimental regulation: 1) Forskolin (a labdane diterpene produced in the Indian Coleus plant) activates AC - binds to AC 2) Cholera toxin causes ADP ribosylation of Gas - therefore a unit unable to associate with By - Cholera toxin binds to the alpha subunit, it causes constant stimulation and activity of the Gs-protein, and this leads to water loss in diarreaha (turns on sodium chloride channels in the gut lumen) - permenant activity of AC It covalently adds a molecule of ADP to the Gs protein. 3) Pertussis toxin inhibits Gi so it remains in its inactive form (GDP bound) - constant activity CAMP Earle Sutherland awarded Noble Prize in 1971 - first to find out about cAMP as a second messenger. CAMP can activate: - Protein kinase A (PKA) - Cyclic-nucleotide gated ion channels - directly activate - Exchange proteins activated by cAMP (EPACS) - switch on a set of kinases CAMP decomposition - so there is no build-up of second messengers CAMP phosphodiesterases CAMP phoshodiesterases breaks the phosphate cycle produce AMP from cAMP. AMP is inactive In cAMP, the phosphate group is linked back 2 carbons in a loop. CAMP AMP Experimental manipulation: Phosphodiesterase inhibitors prevent the break down of cAMP to AMP (e.g Caffeine, IBMX and theophylline) Stimulants - theophylline is used to relax the smooth muscle (CAMP causes smooth muscle relaxation). Bucladesine is a cAMP analog, which mimics cAMP and acts as Phosphodiesterase inhibitor Protein Kinase A (PKA) PKA is a