GPCRs 1 Flashcards
GPCRs
G-Protein Coupled Receptors
Present in all eukaryotes
Involved in most biological responses taste / smell / sight neuronal function inflammation cell growth and homeostasis
Wide variety of different ligands lipids peptides neurotransmitters chemicals light
Low sequence conservation across the family – makes identification in the genome more difficult
> 800 different GPCR genes in humans (approx. 4 % of human genes)
Second messengers
Small molecules that relay signals form cell receptors to intracellular targets.
3 main types:
- hydrophobic: diacyl glycerol (DAG), phosphatidylinositols (PIP3)
- hydrophilic: cAMP, IP3, Ca2+
- gases: nitric oxide (NO)
Levels increase in response to the activation of appropriate receptors:
this can be due to:
- increased synthesis (eg cAMP, IP3, DAG, PIP3, NO)
- or release from intracellular stores (eg Ca2+)
They act by binding to their target proteins and changing the activity of these proteins.
Their increase is transient and they are actively degraded or removed.
GPCR Structure
GPCRs consist of 7 transmembrane a-helix segments; also referred to a 7 transmembrane (7-TM) domain receptors. Extra- and intra-cellular loops N-terminal extracellular tail C-terminal cytoplasmic tail GPCRs do not have any catalytic domains
GPCR activation
GPCRs are thought to exist in either an inactive or active state
Ligand binding affects the equilibrium between these two states
No ligand; inactive state favored.
Agonist: equilibrium shifted towards active state.
Antagonist / inverse agonist: equilibrium shifted towards inactive state.
Neutral antagonist does not shift equilibrium but may block agonist binding.
Structures of GPCRs bound to agonists and antagonists are very similar
Agonist binding is though to disrupt hydrogen bonding in the GCPR
This allows a shift in the orientation of the VI and VII helical segments and the insertion of part of the Gs subunit into the GPCR helical bundle
The molecular details of this are likely to vary between different GPCRs
Heterotrimeric G proteins
Have alpha (α), beta (β) and gamma (γ) subunits bound to the GPCR
G beta/G gamma dimers:
Always found as a dimer of a Gb and a Gg subunit.
Most combinations of the different Gb and Gg isoforms are possible; the functional consequences of these different types of Gb/Gg dimers are not clear.
The C-terminus of the Gg is prenylated – results in the localization of the Gb/Gg dimer to the plasma membrane.
Two main functions:
1. Regulation of the Ga subunit
Promotes the retention of GDP in the Ga subunit.
Helps localize the Ga subunit to the membrane
2. Ga independent signaling
Activation of GIRKs (G protein-coupled inwardly-rectifying potassium channels)
G alpha subunits:
16 genes; at least 23 distinct Ga protein subunits in humans.
Responsible for the majority of signaling activated by heterotrimeric G proteins.
Ga subunits can be myristoylated , helps localize them to the membrane.
Bind to various downstream effectors; the binding site for the effector proteins is similar to that for the Gb/Gg dimer so they cannot both bind at once.
Process intrinsic GTPase activity; hydrolysis of GTP to GDP inactivates the Ga subunit and allows it to reassociate with the Gb/Gg dimer.
Some effectors have GAP activity (eg PLCb). GTP hydrolysis can also be promoted by “regulator of G protein signaling” (RGS) proteins. These also have GAP activity and function to attenuate Ga signaling
cAMP signaling
cAMP is acyclic nucleotide found in organisms
Good molecule as a 2nd messenger as:
- Levels can be increased and decreased rapidly
- Small and diffusible
- Very close control on its intracellular concentration
cAMP is generated by the action of adenylate cyclases and can be removed by phosphodiesterases
Adenylate cyclases
Found in all organisms, generates cAMP
6 classes have been identified:
- Class I, II, IV, V and VI are bacterial enzymes
- Class III adenylate cyclases have been found in both prokaryotes and eukaryotes
Mammalian cells have 10 class III adenylate cyclases:
- 1 – 9 (I-XI) are transmembrane proteins
- 10 (X) is a soluble protein; not regulated by GPCR signaling
Effects of cAMP:
cAMP can activate 2 main cellular pathways mediated by either PKA or EPAC.
PKA has protein kinase activity, EPAC has GEF activity.
Both bind and are activated by cAMP.
Synthetic analogues of cAMP have been described that activate either PKA or EPAC but not both – allows the specific effects of activating PKA or EPAC to be studied.
PKA
Protein Kinase A; member of the AGC family of protein kinases.
Inactive PKA exists as a complex of 2 catalytic and 2 regulatory subunits.
The regulatory subunit contains a pseudosubstrate motif that binds the active site of the kinase subunit.
PKA phosphorylates proteins at a Arg-Arg-Xaa-Ser motif
Transcriptional regulation by PKA
CREB is a member of the bZip transcription factor family
Binds to cyclic-AMP regulatory elements (CRE) in gene promoters
At most target promoters is constitutively bound as a dimer.
In response to elevations in cAMP, PKA is activated and phosphorylates CREB on Ser133
other pathways can also promote CREB Ser133 phosphorylation including Ca2+ and MAPK pathways
Ser133 phosphorylation creates a binding site for the coactivator proteins CBP (Creb Binding Protein) or p300.
CBP and p300 are closely related proteins that can both interact with the RNA transcriptional machinery and acetylate histone to modulate chromatin structure
CREB can be regulated by either Ser133 phosphorylation or CRTC recruitment
The relative importance of these tow mechanisms is both promoter specific and stimuli specific.
The ability of multiple pathways to promote CREB phosphorylation but only PKA to activate CRTCs allows PKA to modulate the gene expression in synergy with other pathways
Termination of cAMP signals
cAMP is removed by phosphodiesterases (PDEs) which hydrolyze cAMP to AMP
11 main families of PDEs;
Some hydrolyze cAMP
Some hydrolyze cGMP
Some hydrolyze both cAMP and cGMP
All contain a C-terminal catalytic domain
Can be regulated at multiple levels: Expression Subcellular localization Protein-protein interaction phosphorylation
Targeting of PKA
PKA can interact with AKAPs (A-kinase anchoring proteins)
Many different AKAPs have been described. All contain a amphipathic helix that binds to the regulatory subunit of PKA
Apart from their ability to bind PKA, different AKAPs show a wide variety of structural diversity and mechanism of action.
APAPs can act by:
Localizing PKA to distinct subcellular compartments
Recruiting PKA to other signaling proteins
mAKAP anchors PKA and PDE to the perinuclear membrane in myocytes; controls both localization and feedback inhibition of the cAMP signal
