G-Proteins Flashcards
G-proteins full name
guanine nucleotide binding regulatory proteins
What are G-proteins?
they are the go-between proteins and act as transducers between receptors and effectors
Two classes of G-proteins have been described
- heterotrimeric G-proteins (large proteins), membrane proteins, consist of alpha, beta and gamma subunits
- monomeric G-proteins (small proteins), not membrane-associated, but bind GTP and GDP
The structure of heterotrimeric G-protein
- consists of alpha, beta and gamma subunits
- in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP (guanosine diphosphate)
- beta and gamma units are tightly bound with each other
G-proteins - the switch between active and inactive states
- G-proteins are membrane proteins, anchored to the membrane through a fatty acid chain
- in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP
- agonist binding to a receptor causes a conformational change which encourages the receptor to bind to the G-protein complex
- this coupling causes the GDP bound to the alpha subunit to be exchanged for GTP
- the G-alpha(GTP) subunit dissociates from the receptor and from the beta and gamma subunits and interacts with the target protein, leading to the generation of second messengers
- in the meantime, the alpha subunit constitutive GTPase hydrolyses GTP to GDP
- this hydrolysis process is the turn-off signal that induces G-alpha to disengage from the effectors
- finally the G-alpha-GDP subunit reunites with the beta and gamma subunits
Summary of G protein activation
G-proteins are membrane proteins comprised of 3 subunits: alpha, beta and gamma
- in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP (GDP bound G-alpha))
- agonist-receptor binding causes receptor conformational change, which encourages the receptor to bind to the G-protein complex
- the receptor-G-protein coupling promotes the exchange of GDP for GTP on the G-alpha subunits
- the G-alpha(GTP) dissociates from the receptor and from beta and gamma subunits, and interacts with the target protein (e.g. adenylyl cyclase), leading to the generation of second messengers
- the cycle is completed by the hydrolysis of GTP to GDP by the G-alpha constitutive GTPase, resulting in the re-association of G-alpha(GDP) with the beta and gamma subunits
GPCR signalling cascade
receptor (sensory receptors, peptide receptors, hormone receptors, neurotransmitter receptors) –> G-protein –> effector (enzymes - adenylyl cyclase, phospholipase A, phospholipase C; ion channels) –> 2nd messenger (cyclic nucleotides, products of inositol lipids, calcium) –> 2nd effector (enzymes - kinases, phophatases; ion channels)
The main G-proteins subtypes and their functions
- G-alpha-s
G-alpha-s
Associated receptors
- many amine and other receptors (e.g. catecholamines, histamine, serotonin)
Main effectors
- stimulates adenylyl cyclase, causing increased cAMP formation
Notes
- activated by cholera toxin, which blocks GTPase activity, thus preventing inactivation
The main G-proteins subtypes and their functions
- G-alpha-i
G-alpha-i
Associated receptors
- as for G-alpha-s, also opioid, cannabinoid receptors
Main effectors
- inhibits adenylyl cyclase, decreasing cAMP formation
Notes
- blocked by pertussis toxin, which prevents dissociation of alpa-beta-gamma complex
The main G-proteins subtypes and their functions
- G-alpha-o
G-alpha-o Associated receptors - as for G-alpha-s, also opioid, cannabinoid receptors Main effectors - limited effects of alpha-subunit (effects mainly due to beta-gamma subunits) Notes - blocked by pertussis toxin - occurs mainly in nervous system
The main G-proteins subtypes and their functions
- G-alpha-q
G-alpha-q
Associated receptors
- amine, peptide and prostanoid receptors
Main effectors
- activates phospholipase C, increasing production of second messengers inositol triphosphate and diacylglycerol
Characteristics of GPCR and G-protein interactions
General
- Multiple coupling of receptors to different G-protein families
- Divergence of signal
- Bidirectional control of target enzyme
- Convergence of signals
- Specificity of receptor-G-protein interactions
Characteristics of GPCR and G-protein interactions
- Multiple coupling of receptors to different G-protein families
- Multiple coupling of receptors to different G-protein families
e.g. Beta-ARs couple to both Gs (classical) and Gi proteins
Gs-mediated activation if PKA causes phosphorylation of beta-AR which then switches the receptor coupling to Gi and simulates the mitogen-activated protein (MAP) kinase
see PKA signalling pathway
Characteristics of GPCR and G-protein interactions
- Divergence of signal
- Divergence of signal: Both alpha and beta-gamma subunits are signalling mediators
The specific functions of the beta-gamma complex are not yet known, but it may be involved in:
- activating potassium channels
- inhibiting voltage-gated calcium channels
- activating GPCR kinases
- activating mitogen-activated protein kinase cascade
Characteristics of GPCR and G-protein interactions
- Bidirectional control of a target enzyme
- Bidirectional control of a target enzyme
- the heterogeneity of G-proteins allows different receptors to exert opposite effects on a target enzyme (e.g. adenylyl cyclase)
- in the intestine and bladder, M3 receptor activation causes smooth muscle contraction
- the ratio of M2:M3 = 70:30
- M2 receptor doesn’t seem to play a role in the control of smooth muscle activity
- what is the function of M2 receptor in the intestine and bladder?
- see diagram
- M2 receptor inhibits cAMP formation, thus inhibiting cAMP-dependent relaxation of smooth muscle
Characteristics of GPCR and G-protein interactions
- Convergence of signals
- Convergence of signals: different receptors act on the same family of G-proteins
e. g. alpha-2-adrenergic, adenosine A1, muscarinic M2, opioid receptors stimiulate G-alpha-i/o, which leads to:
- inhibition of AC
- activation of K channel (via G-alpha-i and or/ beta-gamma)
- inhibition of calcium channels (via Go)
Characteristics of GPCR and G-protein interactions
- Specificity of receptor-G protein interactions
- Specificity of receptor-G-protein interactions
- Hundreds of GPCRs use a family limited range of G-proteins.
- How do they get specificity?
- Studies by antisense oligos-directed against G-alpha, beta, and gamma subunits show that the selectivity of receptor-effector coupling is determined by the specific combination of alpa, beta, and gamma heterotrimers
- e.g. carbachol acts on M1 muscarinic receptor in basophilic lymphocytes to stimulate PLC via an alpha-q/11-beta-1/4-gamma-4 complex
Small GTPases (small G-proteins)
- monomeric G-protein
- also called the Ras GTPase
- more than a hundred proteins in this superfamily
- can loosely be divided into: Ras, Rab, Arf, Ran, Rheb, Rho and Rap families
- they regulate a wide variety of processes in cells, including growth, cellular differentiation and proliferation, cell survival, angiogenesis, vesicle transport, hypertrophy and cancer etc.
- G-alpha-12/13 couples to the activation of Rho (see diagram)
Diseases associated with G-protein mutations
- Albright hereditary osteodystrophy (AHO) - Gs loss
- Pseudohypoparathyroidism type 1a - Gs loss
- Testotoxicosis - Gs gain/loss
- McCune Albright syndrome - Gs gain (mosaic)
- Somatotroph adenomas with acromegaly - Gs gain
- Ovarian and adrenocortical tumours - Gi gain
Diseases associated with G-protein mutations
- Ovarian and adrenocortical tumours
Ovarian and adrenocortical tumours
- G-alpha-i subunit mutations are present in 300% of ovarian sex cord tumours and in adrenocortical tumours
- activating G-alpha-i mutations result in constitutively activated signal transduction; this may lead to tumourigenesis
Diseases associated with G-protein mutations
- McCune Albright syndrome
- G-alpha-s mutations occur in either Arg201 or Gln227 residues
- the mutations disrupt the intrinsic GTPase activity, thus the G-alpha-s gets stuck on the “on” position
- if the mutation affects the skin cells, it causes darker than normal pigment (mosaic)
- if the mutation affects bone cells, it causes weakness and fractures
- in hormone-producing cells, the mutation causes excess release of hormones
Diseases associated with G-protein mutations
- Albright hereditary osteodystrophy (AHO)
Patients who inherit a heterozygous G-alpha-s mutation develop AHO, a syndrome characterised by one or more of these clinical features:
- short stature, subcutaneous ossifications, centripetal obesity, depressed nasal bridge, and mental or developmental retardation
Diseases associated with G-protein mutations
- Pseudohypoparathyroidism type 1a
- in addition to the AHO phenotype, patients who maternally inherit G-alpha-s mutations also develop resistance to various hormones that stimulate G-alpha-s/cAMP in their target tissues
- in contrast, patients who paternally inherit G-alpha-s mutations develop only the AHO phenotype
Rho-GTPases and cancer
Over expression, rearrangement, point mutations or alternative splicing at RHO proteins can cause a variety of different types of tumours/cancers
see Rho signalling pathway
Rho GTPases as therapeutic targets in cancer
Different strategies to interfere with Rho-GTPase signalling pathway
- Rho-spatial regulation
- Rho-GEF interaction inhibition
- Rho-nucleotide interaction inhibition
- Effector inhibitors
- cell migration, cell cycle, apoptosis, gene transcription
Common drugs used in large G-protein research
- GTP/GDP analogs: Gpp[NH]p, GTP-gamma-S
- Pertussis toxin (PTX): catalyses the ADP-ribosylation of the Gi, Go alpha subunits which interferes GDP exchange for GTP
- Cholera toxin (CTX)-induces ADP-ribosylation of an Arg residue on G-alpha-s-protein and inhibits GTPase activity, resulting in persistent G-protein activation
