Cyclic AMP Flashcards
What GPCRs increase adenylyl cylase activity?
Beta 1,2,3
D1, D5
What GPCRs decrease adenylyl cyclase activity?
M2,4
Alpha 2
GABAb
What can cAMP go on to modulate?
Cyclic nucleotide regulated GEFs
PKA
Cyclone nucleotide gated channels
How many isoforms of AC are there?
10 but only 1-9 are membrane bound
What isoform does G(alpha)i actually increase AC activity?
AC2
Which isoform is activtated by calcium calmodulin?
AC1
Which isoform is inhibited by calcium?
AC5
Structure of AC?
Intracellular N terminus M1 (consists of 6 TMDs) Intracellular C1 (forms catalytic domain) M2 (consists of 6 TMDs) C2 (forms catalytic domain) Intracellular C terminus
What are the importance of C1 and C2 domains?
Both are homologous domains with catalytic activity and site of enzymes
What regulates AC activity?
The Ga subunit
Sometimes calcium
PKA and PKC
Why are different regulatory processes and numerous isoforms of AC and PDE’s important?
Cell is continually interpreting signals and this allows for cross talk between different pathways
Cells specific responce to extracellular signals depend on isoform they have
What does Gai, BY and Gas bind to on AC?
Gai - C1
BY - C1 + 2
Gas - C1 + 2
How many families of phosphodiesterase are there?
11
Which PDE are specific for degrading cGMP?
5,6 and 9
Which PDE are specific for degrading cAMP?
4,7 and 8
What PDE are non specific for the cyclic nucleotides?
1,2,3,10 and 11
Where does PKA phosphorylate?
Serine and theonine residues
However, it also recognises proximal amino acids and distal aminoacids ‘consensus recognition sequence’. This sequences is essential for phosphorylation to occur
What is the structure of inactive type 1 PKA?
2 regulatory subunits (4 isoforms exist) and 2 catalytic subunits (3 isoforms exist (alpha, beta and gamma))
Why is the benefit of a number of isoforms for the regulatory and Catalytic subunits?
Can form different hetromultimers
Different patterns of expression and sub-cellular localisation in involved in different cellular processes
How is type 1 PKA activated?
4 Camps binds to regulatory subunit (forming the regulatory subunit dimer) of PKA which causes the release of two catalytic subunit monomers
What is the effect of phosphorylation of proteins?
Changes activity of proteins
Changes affinity for associated proteins which can regulate subcellular location
What is PKA type 2?
The regulatory subunit is bound to A kinase anchoring protein (AKAP) which helps for a protein complex between PKA and it substrate. This is localised to the cellular target of the substrate
How is PKA 2 activated?
4 cAMPs bind to regulatory subunit which opens up the catalytic subunit to phosphorylate the substrate
What is the advantage of AKAP bound PKA?
Everything is localised which allows for quick, efficient and specific phosphorylation
Name some examples of PKA targets
NT synthesis
In heart will increase ionotropic and chronotropy
Ion channels (AMPA at s845)
Regulation of enzymes and proteins
What does DARPP-32 stand for?
Dopamine and cAMP regulated phosphoprotein of 32KDa
In the DARPP-32 system what effect does PKA have?
Phosphorylated DARPP-32 at Threonine 34 which inhibits protein phosphatase 1
PKA activates protein phosphatase 2A which dephosphorylates DARPP-32 at theonine 75. T75 phospho DARPP-32 inhibits PKA thus PKA attenuates PKA inhibition
This promotes kinase activity over phosphatase activity
What phosphorylates DARPP-32 at tyrosine 75?
CDK5
IN the DARPP-32 system what activates PKA?
Activation of the adenosine A2A receptors increases Gas and increases cAMP
What is the effect of caffeine on the DARP-32 system?
Inhibits the adenosine receptors in the nucleus accumbens
Therefore, DARPP-32 is not phospho at T34 and pp1 is not inhibited
Also, PP2A not activated which leaves T75 phospho DARPP-32 to inhibit PKA
Phosphatase activity > Kinases activity
What are cyclic nucleotide gated channels? Where might they be found?
Intracellular Cyclic nucleotides regulate channel opening
E.g. In the pacemaker cells in heart
Retinal rod cells (cGMP)
Structurally similar to VG K+ channels
What does EPAC stand for?
Exchange protein directly activated by cAMP
How many EPAC isofroms are there?
2
EPAC 1 - mRNA found extensively
EPAC 2 - mainly in brain
What is the function of EPACs?
Have guanine nucleotide exchange factor properties which activates small GTPases such as RAP1 these then go on to signal cell adhesion via intergrins
How does cAMP activate EPAC?
EPAC N ermines contains regulatory domain which acts to autoinhibit the catalytic domain (closest to C terminus)
CAMP binds to the regulatory domain to causes a confirmational change which releases EPAC from auto inhibition (independent of PKA)
List two functions of EPACs?
Role depends on cell type
BetaAR in heart = regulation of cardiac myocyte hypertrophy (RNAi KO of EPAC = hypertrophy)
GLP-1R in pancreas regulates insulin secretion target for hypoglycaemic agents
What are the various way a cell creates a micro domain?
Calveolae
Lipid rafts
AKAP (AKAP-79 binds B2AR to PKA)
Localised PDE - restricts diffusion of cAMP which leads to localised response
Different isoforms of proteins - regulated differently and localised to different compartment. Responce of cell dictated by what isoform it expresses
Within cardiac muscle, what does PKA do?
Phosphorylates L type ca2+ channels (Increases activity)
Phosphorylates Ryanodine receptor. PKA is localised to SER (increases channel conductance)
Phospho of tropnin I (increases calcium sensitivity and increases rate of relaxation)
Phospho of BAR (desensitisation - neg feedback mechanism)
Phosphorylates phospholamban which increases SERCA2 activity)
Why is the increase in cAMP localised to near the SER in cardiac muscle?
Surrounded by PDE which attenuate global cAMP signal
