Define the physiological concept “G protein”.
A nucleotide regulatory binding protein which has inherent GTPase activity and is a signal transducer.
Classify G proteins into two main groups and briefly describe the physiological function of these two groups.
- Monomeric G proteins - growth factor signal
cascades, vesicle fusion, and regulation of actin
cytoskeleton - Heterotrimeric G proteins - signal transducers
Identify (at least) 5 families of heterotrimeric G proteins.
- Gs - the alpha sub-unit activates adenyl cyclase
- Gi - mediators of hormonal inhibition of adenyl
cyclase - Gq - involved in the phospholipase C-DAG-IP3 signal
transduction pathway - Gt (transducin) - major retinal G protein that activates
cGMP phopsphodiesterase - Ga - hydrolyses GTP to GDP + Pi
Name the subunits (with their respective functions) of a typical heterotrimeric G protein.
Alpha subunit - the separated alpha subunit brings about many biological effects such as the activation of phospholipase C and adenyl cyclase.
Beta-gamma sub-unit - modulate the activity of the alpha subunit.
Briefly discuss the physiological importance of G proteins.
They act as signal transducers which convert a sensory, neural or ligand-mediated message (signal or stimulus) into an action potential or an activated signaling pathway in a sensory receptor or cell-specific receptor bearing target cell.
Name factors that influence G-protein function (regulation/control).
1. Helper proteins: Most G (GTP-binding) proteins depend on helper proteins, e.g. GAPs (GTPase Activating Proteins) promote GTP hydrolysis
- Guanine nucleotide exchange factors:
Promote GDP/GTP exchange, example
* an activated receptor
* other receptor-independent activating proteins
Briefly describe the common structural characteristics of serpentine receptors.
- Amino terminal outside the cell: modified by different
N-coupled oligosaccharides; binding of negatively
charged ligands, ligand-binding domain, often heavily
glycosylated - Hydrophobic alpha-helices: hydrophobic clusters of
amino acid residues that form hydrophobic pockets
into which small ligands fit. Amino acid residues in the
third cytoplasmic loop of the alpha helices interact
with G proteins - Cytoplasmic domain: consists of an intracellular
carboxyl terminal that contains sites where
phosphorylation can occur
List 5 examples of ligands that interact with G protein-coupled (i.e. serpentine) receptors.
- Neurotransmitters (e.g. adrenaline, noradrenaline and
dopamine, adenosine and opioids) - Peptides (e.g. glucagon)
- Glycoprotein hormones (e.g. FSH, LH, AND TSH)
- Arachidonic acid derivatives (e.g. prostaglandins)
NAP, G
List 4 specific examples of serpentine receptors.
- A1
- A2a
- A2b
- A3
Indicate the respective broad/ general functional roles of the ligand, the receptor, the G protein and the enzymes in these signal pathways.
Ligand - signal
Receptor - signal discriminator
G protein - signal transducer
Enzymes - signal amplification
List 4 membrane enzymes that can be activated in association with these pathways.
- Adenyl cyclase
- Guanyl cyclase
- Phosphodiesterase
- Phospholipase C
The G APP
State the general function of protein kinases.
They catalyse the phosphorylation of intracellular functional proteins.
Name and briefly describe the three clinically most important G protein-coupled receptor-associated signal pathways.
- Adenyl cyclase-cAMP system:
- cAMP activates protein kinase A
- a beta adrenergic receptor is coupled to a stimulatory
Gs protein which activates adenyl cyclase and
increases cAMP levels
- the alpha2 receptor is always coupled to an inhibitory
Gi protein, which inhibits adenyl cyclase and
decreases cAMP levels - Phospholipase C-glycerol-Ca++ system:
- phospholipase is activated
- PLC splits PIP2 into IP3 and DAG, which act as second
messengers
- IP3 binds to an IP3 receptor on the SR membrane and
and causes Ca++ release from the SR
- DAG and Ca++ additively activate protein kinase C
which catalyses protein phosphorylation on the serine
and threonine residue, thus eliciting a response - Ca++- calmodulin system
- The Ca-calmodulin complex can activate kinase II,
which causes phosphorylation of serine and threonine
residues
- enzymes modulated are myosin light chain kinase,
phosphodiesterases, and phosphorylase kinase
- intracellular Ca content is restored by the Ca++/H+
ATPase pump (one Ca++, 2 H+)
CAP!!!
Describe the 10 steps of activation and mechanism of action of the cAMP second messenger system and the consequences of cAMP formation.
- Ligand interacts w/ serpentine (7-helix) GPCR
- Activated receptor subject to conformational (3D)
change - Conformational in associated G protein occurs
- Nucleotide-binding site on alpha sub-unit of G
protein becomes more accessible to cytosol, where
the GTP [ ] is higher than the GDP [ ] - Alpha subunit of the G protein releases GDP which is
replaced by GTP - Substitution of GDP by GTP causes another
conformational change in the alpha subunit of the G
protein - The alpha subunit of the G protein dissociates from
the inhibitory beta-gamma complex - The dissociated (energized) alpha subunit of the G
protein can now move away to interact w/ and
activate adenylate cyclase by phosphorylating it - Adenylate cyclase can now catalyse the breakdown
of ATP to form the second messenger, cAMP - cAMP activates protein kinase A, which catalyses
the phosphorylation of various functional cellular
proteins, which alters their activity and usually
activates them by adding a high energy phosphate
group
Name the respective functions of adenyl cyclase and phosphodiesterase in signal pathways.
Adenyl cyclase: catalyses the formation of cAMP from ATP
Phosphodiesterase: catalyses the conversion of cAMP to physiologically inactive 5’AMP
Name the direct breakdown products of PIP2 hydrolysis.
IP3 (Inositol triphosphate) and DAG (diacylglycerol)
Identify the receptor that closely resembles the IP3 receptor.
Ryanodine receptor
Briefly describe the two functions of IP3 in this signal pathway.
- Acts as a second messenger
2. Interacts with a sarcoplasmic reticulum IP3 receptor to cause the release of CA++ from the smooth-surfaced ER
Identify the substance that is the lipid portion of DAG and identify one group of local hormones (parahormones) of which this substance is the precursor.
Lipid portion: Arachidonate
Local hormones group(s): prostaglandins, leukotrienes
Identify the enzyme that is activated by DAG.
Protein kinase C
Identify the main function of DAG.
It acts as a second messenger.
Name the 5 main steps in the calcium-calmodulin signal transduction pathway.
- Calcium entry may be initiated by changes in the membrane potential that open calcium channels or a hormone interacting with membrane receptors that open calcium channels
- On entering a cell, calcium ions bind with the protein calmodulin, which has four calcium sites
- When three or four of these sites have bound with calcium, calmodulin changes its shape and initiates multiple effects inside the cell; including activation or inhibition of protein kinases
- Activation of calmodulin-dependent protein kinase causes, via phosphorylation, activation or inhibition of proteins involved in cell’s response to the hormone
- One specific function of calmodulin is to activate myosin light chain kinase, which acts directly on myosin of smooth muscle to cause smooth muscle contraction
Identify mechanisms of signal termination in G protein-associated signaling pathways (mechanisms applicable to all three signal pathways and mechanisms that are specific to each pathway).
Mechanisms applicable to all three signal pathways:
* Ga hydrolyses GTP to GDP + Pi (GTPase)
- presence of GDP on Ga causes it to rebind to
inhibitory beta-gamma complex; adenylate complex
activated
- Receptor desensitisation (varies according to which
hormone has activated the receptor) - Dephosphorylation due to phosphates
Mechanisms applicable to each specific signal pathway:
* Adenyl cyclase-cAMP system
- cyclic nucleotide phosphodiesterases degrade cAMP
to 5’-AMP
- Most cells also possess a mechanism for facilitated
cAMP extrusion
- Phospholipase C-diaglycerol- Ca++ system
- degradation by DAG lipase and enzymatic recycling to
form phosphatidylinositol - sequential IP3 dephosphorylation by enzyme-
catalysed hydrolysis yields inositol, a substrate for
synthesis of phosphatidylinositol - Ca++-calmodulin system
- active Ca++ transport into intracellular compartments
and Ca++ extrusion by plasma membrane-bound Ca++
pumping ATPases deactivates pathway
Identify two pharmacological or exogenous substances that inhibit phosphodiesterase.
- Caffeine
2. Theophylline
Identify two bacterial toxins that disrupt G protein reaction.
- Vibrio cholera
2. Bordetella pertussis
Draw a difference between the commencement of action of neurotransmitters and exogenous/endogenous hydrophilic endocrine hormones that interact w/ ligand-gated ion channel associated receptors, tyrosine kinase receptors and G-protein coupled receptors.
Hydrophilic ligands have a more rapid commencement and shorter duration of action than the lipophilic or amphophilic ligands.
- ligands that interact with ligand-gated ion channel associated receptors have a more rapid commencement and shorter duration of action than ligands that react with tyrosine kinase receptors
- Serpentine receptors are the slowest
