W10L1 Flashcards
Calmodulin and CAM Kinase II
Proteins regulated by Ca2+ include PKC and calmodulin, which has no intrinsic enzyme activity but can bind to and modulate multiple target proteins in a Ca2+–dependent manner
- calmodulin has 4 calcium binding domains
- affinity of calmodulin increases for target proteins when it is bound to calcium
CaM-kinase II (calmodulin-dependent kinase II) is switched to its activated state when exposed to Ca2+ / calmodulin; remains activated even after cytosolic Ca2+ levels return to normal
- Autophosphorylation prolongs duration of activation of CaM-kinase II beyond duration of activating Ca2+ signal
- is called a CaM-kinase II “memory switch”
- CaM-kinase II remains active until a phosphatase comes along and removes the phosphate from CaM-kinase II, putting it back into inactive form
Modulation of G protein signaling by intracellular proteins
- Some effectors can deactivate the G proteins they interact with
- RGS proteins deactivate G proteins
- Phosducins bind to Gbg and limit heterotrimer
formation - GDIs for heterotrimeric G proteins (e.g., GPSM3/AGS4) inhibit GDP dissociation while blocking Gbg binding to Ga
- non-receptor GEFs (e.g., Ric-8) can activate Ga
- GPCRs can be phosphorylated by various kinases (GRKs, PKA, PKC), which in many cases decreases their ability to signal through G proteins
- Arrestins impede Ga binding, promote receptor internalization and can modify signaling functions
- Anchoring/scaffolding proteins bring together signaling proteins to enhance efficiency
Reciprocal regulation between G proteins and effectors
Phospholipase Cb (C beta) is a GTPase accelerating protein(GAP) for Gaq. Thus Gaq promotes the ability of PLCb (beta) to hydrolyze PIP2 while PLCb promotes the ability of Gaq (alpha q) to hydrolyze GTP
p115RhoGEF and LARG are GAPs for Ga12 and Ga13
some effectors limit their own activation
RGS proteins (Regulator of G Protein Signaling proteins)
- RGS proteins counter the effects of GPCRs
- 20 different RGS gene products, plus splice variants
– vary widely in size and structure (20 kDa – 160 kDa)
– all contain ~120 aa RGS domain that binds to Ga - RGS proteins are GAPs that bind to G proteins and
increase the rate at which GTP is hydrolyzed - Can produce other inhibitory effects on GPCR signaling
- Also can bind to GPCRs, effectors, and auxiliary proteins to modulate signaling
- Known to play an essential role in some signaling pathways
- Can be activated or upregulated to limit G protein-mediated signaling
RGS protein subfamilies
R4, RZ, R7, R12
R4 and RZ subfamilies contain mostly small proteins made up of an RGS domain plus short N- and C-termini
R7 family contains RGS 6, RGS 7, RGS9, RGS11
- R7 subfamily RGS proteins contain two additional domains
– the GGL (Ggamma-like) domain binds to Gbeta5, and both proteins are unstable unless bound to one another
– the DEP (Disheveled/EGL-10/Plextrinhomology) domain is a module of about 100 amino acids which serves as a binding interface for membrane anchoring proteins (R9AP, R7BP)
R12 subfamily RGS proteins RGS12 and RGS14 contain a conserved GDI motif that inhibits GDP dissociation from isolated Galpha. This prevents G protein from being activated - it locks it into the inactive GDP bound state, and impedes the binding of GTP. It is unclear what effect this has on GPCR signaling.
There are no RGS proteins that are GTPase accelerator proteins for Gas
- intrinsic GTPase activity in Gas is faster than that of the rest, so there is less of a need for a second protein to turn it off
Biochemical properties of RGS proteins
conserved 15kDa RGS domain binds to Ga at switch regions; other RGS protein features highly variable
RGS bound to a ‘GTP bound Galpha protein’ will increase rate of Ga GTP hydrolysis
– ~100x GTPase rate of free Ga-GTP
– limited selectivity among G proteins
– ~1000x GTPase rate of GPCR-G-GTP
“effector antagonism” - may compete with or hinder G binding to effectors
other RGS binding partners include GPCRs, effectors and scaffolding/anchoring proteins
– GPCRs may target RGS proteins to G proteins
– effector activities can be directly modified by the binding of RGS proteins
Larger RGS proteins (R7, R12 subfamilies) have additional domains that confer other functions
Physiological roles of RGS proteins
- desensitization – expression of some RGS proteins is induced by 2nd messengers –> decreased receptor response
- maintaining rapid signaling kinetics – inwardly rectifying potassium (GIRK) channels , visual system
- muting or dimming of receptor signals (RGS-induced decreases in agonist sensitivity and/or max effect)
- additional functions of non-RGS domains
- selective effects on GPCR signals
– inhibition of G protein signaling by an RGS protein can vary depending on which receptor is activating the G protein
– some RGS proteins bind directly or indirectly to GPCRs
– > RGS proteins can be targeted to receptor- associated G proteins
Inhibiting RGS proteins increases GPCR signaling
–> Agonist potency/efficacy is inversely related to the activity of RGS proteins
RGS proteins are required for normal GIRK channel function
G protein-gated inwardly rectifying potassium (GIRK) channels mediate hyperpolarization of excitable cells in response to GPCR activation (eg., in brain and myocardium)
In recombinant systems (or when expressed artificially), GIRK channel activation (rate of channel opening) and deactivation (rate of channel closing) kinetics are significantly slower than in physiological systems
- closing of channel corresponds to GTP hydrolysis
Normal channel kinetics can be restored by the expression of RGS proteins
RGS effect on opening implies an action in addition to GAP activity
Opening of channel is also faster in presence of RGS protein
RGS9 is required for normal mammalian vision
Proper temporal resolution of the light response in retinal photoreceptors requires the rapid inactivation of the G protein transducin by RGS9.
The activity of RGS9 is decreased under dim light and increased by bright light
There is a slowed recovery of the rod photoresponse in mice lacking RGS9
In humans with mutations in RGS9 or its associated anchoring protein (R9AP), the ability to see moving objects is greatly decreased. It is important for visual acuity
Desensitisation
Desensitisation
* The diminution of an agonist’s effect or attenuation of a prolonged sensory input.
Mechanisms include:
– changes to the receptors
– exhaustion of intracellular mediators
– compensatory physiological changes
G protein-coupled receptor desenstitization
Loss of receptor responsiveness as a result of continued stimulation
– physiological examples: reduced ability to detect odours over time, adjusting to bright light/darkness
Development of acute drug tolerance at GPCRs
– at the level of the receptor, responsiveness is progressively decreased as a function of time and drug concentration
– phosphorylation –> internalization –> down regulation
Stepwise process; all steps reduce agonist response
1. phosphorylation: decrease in agonist ability to
activate G protein (seconds to minutes)
2. internalization: removal of receptor from cell surface via endocytosis (minutes)
3. downregulation – decrease in receptor number
(minutes to hours) due to
– increased degradation
– decreased mRNA level –> decreased synthesis of receptor protein
Receptor activation leads to phosphorylation by kinases:
1. 2nd messenger-dependent kinases (GPCRs have phosphorylation sites for PKA and PKC; phosphorylation by PKA and PKC tends to decrease receptor responsiveness by agonist)
* Protein kinase A activated by cAMP
* Protein kinase C activated by DAG + calcium
- G protein coupled receptor kinases (GRKs)
* these phosphorylate agonist activated receptors
* these are conformationally sensitive kinases, so the kinase does not recognize the inactive receptor as a substrate. It is only the active conformation of the receptor that the kinase recognizes and phosphorylates
* Preferentially act on agonist-occupied receptors
Covalent modification of serine residues by kinases decreases ability of receptor to couple to G protein
- this decreases agonist-dependent G protein activation and causes recruitment of “desensitizing” proteins
- Arrestins compete with G protein for receptor
- Arrestins are intracellular proteins that are scaffolding proteins. They bind on the same interface on the receptor where the G protein binds. So phosphorylation by GRKs increases the affinity of arrestins and decreases the affinity of G proteins, thereby reducing the ability of the receptor to signal through G proteins
Homologous desensitization and heterologous desensitization
Agonist goes to receptor, causing activated receptor. This leads to 2 types of desensitization pathways:
- Homologous desensitization (specific to the activated receptor itself)
- GRK phosphorylates the activated receptor
- arrestin comes in to create an arrestin-receptor complex
- this results in loss of G-protein coupling and causes endocytosis - Heterologous desensitization (my phosphorylate not only the activated receptor, but may also phosphorylate other receptors that are nearby and increase their activity as well; so not very specific)
- the activated receptor causes activation of PKA, PKC, etc.
- this causes non-specific receptor phosphorylation
- results in reduced G-protein coupling
GRK family
GRKs are serine/threonine kinases, recruited to PM in response to GPCR activation
7 GRK isoforms
- GRK1 & 7 – retina
- GRK4 – testes, kidney and brain
- GRK2, 3, 5 & 6 - ubiquitous
Kinase domain (KD) binds to and phosphorylates activated GPCRs
All GRKs contain a divided RGS homology (RH) domain that binds to Gaq (has little or no
GAP activity)
Some contain extra scaffolding domains:
- Plextrin homology (PH) domain of GRK2, GRK3 near C-terminus; this allows them to bind to Gbg subunit and bring it closer to the receptor
Evidence for phosphorylation-independent attenuation of GPCR signaling
How is GPCR signalling attenuated?
An activated receptor changes its conformation and increases the ability of the GPCR kinase to phosphorylate, leading to phosphorylation of one or more residues. This phosphorylation of the receptor increases the affinity of arrestin and decreases the affinity of the G protein
- activated GPCR stimulates GRK to phosphorylate the GPCR on multiple sites
- arrestin binds to phosphorylated GPCR
- results in desensitized GPCR
Arrestin is a protein that does not have much function on its own other than bind to other proteins
4 arrestin isoforms
* visual arrestin - rod outer segment of photoreceptor cells of retina
* cone arrestin – cone cells of retina in opsin desensitization
* β-arrestin 1 & 2 – ubiquitous, involved in desensitization of all other GPCRs, enriched in immune system and neurons
Mechanisms of GPCR endocytosis
- Ligand binding
- Receptor phosphorylation
- beta-arrestin interaction
- association with clathrin-coated pits
- dynamin-dependent pinching off
- Endocytosis
- β-arrestins, by their association with the AP-2 adaptor complex and clathrin, target GPCRs to clathrin-coated pits
- Clathrin triskelions also bind AP-2
- Internalized receptors are prevented from
binding to extracellular agonist molecules
