W10L2 Flashcards
Arrestin signalling
Arrestin binding to phosphorylated GPCRs results in desensitization of G protein- mediated responses, but it can also initiate signaling via alternative pathways
Arrestins are conformationally flexible and can bind to many different molecules
Arrestins are scaffolding proteins which can link GPCRs to either desensitization or signaling pathways
Arrestin binding to the same receptor phosphorylated by different GRKs (presumably at non-identical residues), can have distinct signaling consequences
Recent studies call into question the ability of GPCR-arrestin complexes to signal independently of G proteins
Recent studies call into question the ability of GPCR-arrestin complexes to signal independently of G proteins:
1. arrestin-dependent ERK phosphorylation was abolished in G protein-depleted cells
- implies that GPCR-arrestin complex is not sufficient to convey signal
2. Recent studies suggests the existence of GPCR-arrestin-G protein complexes
Not clear which previously identified GPCR-arrestin pathways are truly G protein-independent
GPCR-Ga protein selectivity
Activation of somatostatin receptors in pancreatic islet cells limits insulin release in a PTX-sensitive manner
- PTX increases insulin levels and reduces glucose levels
Somatostatin limits insulin release
Basal insulin release is increased in islet cells from Gao2 knockout mice, and inhibitory effect of
somatostatin is lost
Elevation in blood glucose after glucose challenge is reduced by knockout of Gao2 but not Gao1 or Gai
Results imply that SST receptor-mediated inhibition of insulin release requires Gao2, i.e., other Ga subunits cannot substitute
Discrete coupling of GPCRs to Ca2+ channel inhibition
M4 muscarinic and somatostatin receptors both trigger PTX-sensitive inhibition of Ca2+ currents in GH3 cells
- Gb3 (G beta 3) knockdown eliminates response to carbachol (CA) but not somatostatin (SST)
- Gb1 (G beta 1) knockdown selectively eliminates SST response
Carbachol activates muscarinic receptors
Muscarinic receptor couples through a G protein that contains Gb3 (G beta 3)
Somatostatin response is mediated by a G protein that contains Gb1 (G beta 1)
Results imply that these GPCRs do not share G proteins, i.e., M4 and SST signaling pathways are discrete
Intracellular localization of GPCR signals
An increase in cyclic AMP in response to an extracellular signal. This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a G-protein-linked receptor to cause a rapid rise in the intracellular concentration of cyclic AMP.
- To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP.
- Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level.
- In the resting cell, the cyclic AMP level is about 5 × 10-8
- Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10-6 M, an increase of more than twentyfold.
Not all parts of the cell had cyclic AMP, meaning there is localized signalling at specific parts of the cell
Note that not all areas of the cell are affected equally.
Similarly localized GPCR signals are found in other cells.
GPCRs signal through discrete pathways
Many GPCRs are capable of activating >1 type of G protein
Many G proteins are capable of regulating >1 type of effector
Some effectors can be activated by >1 type of G protein
However – much signaling occurs through discrete R-G-E pathways within cells, implying a higher level of organization
Although it is still generally assumed that G proteins shuttle between receptors and effectors, much evidence suggests that GPCRs, G proteins and effectors can come together to form signaling complexes
How is pathway selectivity maintained?
Each cell contains multiple different GPCRs, G proteins, and effectors
Co-expression and reconstitution studies indicate that G proteins interact promiscuously with both GPCRs and effectors
In contrast, studies on endogenous signaling pathways show that these tend to be discrete and that many biochemically possible pathways are “ignored”
Why is GPCR signaling exclusory?
– GPCRs, G proteins and effectors might form into macromolecular complexes
– Elements of the signal transduction machinery may be held in close proximity through binding to the cytoskeleton or scaffolding/anchoring proteins
– Movement of proteins within the membrane may be limited, allowing proteins to interact only with those in the same “membrane compartment”
– Note that these possibilities are not mutually exclusive
Many GPCR signals are coordinated via scaffolding/anchoring proteins
Anchoring serves as a regulatory mechanism that coordinates complex signaling events, both spatially and temporally
- both activation and deactivation can be accelerated by bringing the proteins in close proximity of one another
Points of cytoskeletal attachment
Grouping of proteins that are part of the same signaling pathway –> increased substrate availability
Major types: PDZ domain-containing proteins bind to proteins containing a PDZ ligand motif (usually at the C terminus), AKAPs bind to PKA and its associated signaling proteins
Molecular aberrations disrupting these complexes can be linked to the progression of various disease states
AKAPs (A kinase anchoring proteins)
AKAPs are a family of >50 structurally diverse proteins that contain an amphipathic helix that binds to the amino termini of the PKA regulatory domains (usually RII)
Each AKAP also contains a unique subcellular targeting domain that restricts its location within the cell, and thus confines the PKA holoenzyme to discrete intracellular locations
Other signaling proteins brought into close proximity with PKA via an AKAP may include phosphatases, phosphodiesterases, other kinases, and receptors
AKAP79 aka AKAP5
AKAP79 (aka AKAP5) binds directly to beta2-AR, PKA, PKC, and protein phosphatase 2B, and indirectly to the ionotropic glutamate receptor
- AKAP79 is targeted to the plasma membrane by three NH2-terminal basic domains and is recruited to the AMPA receptor by binding to the membrane-associated guanylate kinase (MAGUK) proteins.
- AKAP79 also associates with b2-adrenergic receptors (b2-AR). This enhances b2-AR-induced cAMP-PKA signaling by recruiting PKA close to the receptor and the site of adenylyl cyclase activation.
- PKA phosphorylation of the b2-AR leads to desensitization of the receptor (and may promote Gi coupling and MAP kinase activation); however, PKA phosphorylation enhances glutamate receptor activity. Thus AKAP79 brings the cAMP-generating machinery, PKA, and the substrates into close proximity.
- In addition to anchor PKA, AKAP79 also recruits protein kinase C (PKC) and protein phosphatase 2B (PP2B) and thereby integrates several signaling pathways into a multiprotein complex.
Bidirectional regulation of signaling by AKAP signaling complexes
One function of AKAPs is to coordinate signaling complexes by recruiting multiple signaling enzymes near potential substrates, effectively joining upstream activators with downstream targets
This mechanism permits the association of an entire signaling complex with a specific substrate
A valuable feature of some AKAP signaling complexes is the presence of signal transduction and signal termination enzymes in the same network, eg., the clustering of protein kinase and phosphatase activities
This creates focal points of enzyme activity where the bidirectional regulation of signaling events can be controlled and the phosphorylation status of target substrates is precisely regulated
This allows the coordination of phosphorylation and dephosphorylation events mediated by the enzymes associated with AKAPs
Negative feedback loop coordinated by mAKAP
The 300-kDa muscle-specific AKAP (mAKAP) associates with PKA,and PDE4D3 (a phosphodiesterase), plus the phosphatases PP1 and PP2A, and the ryanodine receptor
Under basal conditions, PKA is inactive and the PDE maintains low intracellular concentrations of cAMP.
Upon hormonal stimulation, the generation of cAMP increases and overcomes the basal rate of PDE-mediated cAMP degradation, allowing for activation of PKA and phosphorylation of local substrates.
PKA phosphorylation of mAKAP-anchored PDE enhances PDE activity, causes increased cAMP degradation, and results in decreased PKA activity.
PDZ domain-containing proteins
The PDZ (PSD95/DLG/ZO-1) domain is ~100 amino acid residues long and is found in numerous scaffolding proteins.
The PDZ domain binds to proteins containing a short PDZ motif or “ligand” (~ 5 amino acid residues, usually found at the C-terminus)
PDZ domains are involved in the recruitment and interaction of proteins, and aid the formation of signalling networks. Some proteins have multiple PDZ domains and thus can bring together several PDZ ligand-containing proteins.
There are >400 PDZ domain-containing proteins, and at least a dozen of these are able to bind to GPCRs
Some proteins contain both PDZ domains and PDZ ligands, and thus can form extended signaling complexes
PDZ domain-containing proteins associate with the extreme C-terminus of GPCRs
At least 20 GPCRs can bind to PDZ domain-containing proteins via a PDZ “ligand” at the C- terminus
The endothelin ETA receptor
contains an internal PDZ ligand
PDZ ligands are USUALLY found at C-terminus, but not always. They can exist in other places
Invertebrate visual transduction “signalplex”
INAD contains 5 PDZ domains and coordinates the activities of multiple components within this signaling pathway. INAD can also bind to itself
Signalling proteins in fly photoreceptors are assembled into a multimolecular complex by the PDZ domain protein INAD (inactivation no afterpotential D).
The INAD signalling complex includes the ion channel TRP, phospholipase C, the eye-specific protein kinase C, and other proteins
The major INAD ligands are degraded in some INAD mutants, indicating that assembly of the signalling complex is crucial for the stability of its protein components.
