Receptors for what type of molecules are intracellular?
Steroid
Thyroid Hormone
1) Reception
2) __________
3) Response
Transduction
What are the three “superfamilies” of CELL SURFACE receptors?
GPCRs
Ligand-gated ion channels
Enzymatic receptors
Define the term ‘agonist’
Define in terms of affinity and efficacy also
Something that binds to the receptor and ACTIVATES it, leading to signal transduction events.
Agnoists bind with affinity AND efficacy
Define the term ‘antagonist’
Define in terms of affinity and efficacy also
Something that binds to the receptor but DOES NOT activate it, blocking the effects of the agonist at the receptor.
Antagonists possess affinity but do not possess efficacy
GPCRs consist of how many transmembrane domains?
7 TM domains
GPCRs have an extracellular _____ -terminal
N-
GPCRs have an intracellular _____-terminal
C-
Each GPCR has it’s own unique _______ ________ sequence, ranging from _____ - _______ in length
Amino acid
300-1200
How many regions of GPCRs are responsible for ligand binding? What are formed by?
2
Formed by either:
2-3 transmembrane domains
or -N terminal region
An activated GPCR must interact with another protein called a what?
Guanine-nucleotide binding protein (G- protein)
G proteins are made up of how many subunits?
They are therefore be defined as what?
3 subunits
Heterotrimeric
Though made up of 3 subunits, functionally how many subunits does a G protein have?
Two functional subunits
“Dimeric”
What are the three subunits of G proteins and how are they arranged?
Alpha, Beta, Gamma
Alpha is associated to the Beta Gamma subunit.
The alpha subunit dissociates from the beta gamma subunit upon binding of agonist but the beta and gamma subunits do not dissociate.
When an agonist binds to a GPCR what happens in order to generate a downstream signal?
- Agonist binds to GPCR on cell surface
- GPCR exchanges a GTP for a GDP on the alpha subunit
- The alpha, beta, gamma complex dissociates into alpha bound to GTP and beta gamma subunits
- Each of these subunits can then interact with effector proteins
What feature of the receptor, G-protein and the effector increases the efficacy of the effector protein response?
They are all co-localised to the plasma membrane
Most diseases are caused by inefficient ______ of the G-protein signalling
“Turn- off”
How is G-protein signalling terminated?
GTPase activity of the alpha subunit which hydrolyses GTP back to GDP. The alpha-GDP and beta gamma subunits then reform an inactive heterodimeric complex
Is the alpha subunit a good or a poor GTPase?
Does it hydrolyse GTP quickly or slowly?
What does this mean in terms of signalling?
The alpha subunit is a poor GTPase
It hydrolyses GTP slowly
This means that there is a sufficient period of time for the alpha and beta gamma subunits to pass on their signals
What are the three types of G- protein based on the G alpha subunit?
Galphai
Galphas
Galphaq
What are the effector proteins involved with each G protein subtype and are they inhibitory or stimulatory
Galphai inhibits adenylyl cyclase
Galphas stimulates adenylyl cyclase
Galphaq stimulates phospholipase C
What is the acronym to help remember these types of G-protein and their receptors?
Q- alpha 1
I-alpha 2 These are Sympathetic
S-beta 1
S-beta 2
Q- musclarinc 1 These are Parasympathetic
I- musclarinic 2/4
Q- musclarinic 3
Name two toxins that interfere with G protein function
Cholera toxin (CTx) Pertussis toxin (PTx)
How does CTx infer it’s toxicity in the cell?
It modifies all the Gs containing proteins and means that they can no longer hydrolyse GTP. They are therefore permanently switched “on” leading to extremely hightened cAMP production
How does PTx infer it’s toxicity in the cell?
It modifies a Gi protein and does not allow it to be stimulated, it cannot dissociate and is therefore not activated
If some examples of second messengers
cAMP, IP3 and DAG
What is the role of adenylyl cyclase once it is activated in cells?
Converts ATP into cAMP
What is the role of Phospholipase C?
The convert PIP2 into IP3 and DAG
Effectors can be ___________ or ___________
Enzymes or Ion channels
Once cAMP is produced through the activity of adenylyl cyclase, what happens?
cAMP activates Protein Kinase A (PKA)
PKA is a hydro______ molecule that is readily available in what part of the cell?
hydroPHILIC
In the cytoplasm
What is the structure of Protein Kinase A?
Made up of 4 subunits: 2 Regulatory subunits (R) and 2 Catalytic subunits (C)
What is the role of the R subunits in Protein Kinase A?
To regulate the activity of the C subunits
What is the role of the C subunits in Protein Kinase A?
To regulate the catalytic activity of Protein Kinase A (PKA) by phosphorylating target proteins in the cell
What happens when cAMP levels are increased?
They bind to the R subunits and cause the C subunits to dissociate and phosphorylate target proteins in the cell
Phopholipase C converts PIP2 into what?
IP3 nd DAG
What are the basal levels of free Ca2+ in the main extracellular sites?
1mM
10^-3 M
What are the basal levels of free Ca2+ in the main intracellular sites i.e. the “stores”: SER/ER?
1mM
10^-3 M
What are the basal levels of free Ca2+ in the cytosol of cells i.e. the “sink”?
100nM
10^-7 M
________ can also be regarded as an intracellular Ca2+ store but this is more to BUFFER excess Ca2+ than to act as an functional reservoir
Mitochondria
Intracellular stores of Ca2+ reaches _____ concentrations to provide the cell with access to a large reservoir source when required
high
Smooth endoplasmic reticulum (SER) store is known as a rapid or non-rapid release store?
Rapid response
Which Ca2+ store is known as non-rapid release store?
Mitochondria
Calcium is a divalent (or bivalent) meaning what?
It has a valance of two and therefore forms two chemical bonds
Why is calcium’s divalent nature important for its role aa a messenger?
It means that it is attracted to points on biological molecules that are negatively charged or polarised
What is distinctive about the election clouds on calcium?
They are ‘polarisable’ or ‘squishy’ meaning they can be easily distorted- can fit into irregular/enclosed spaces
What is the advantage of the large ionic gradients of calcium between the cytosol, SER/SR/mitochondrial stores and extracellular calcium?
Large changes in the cytosolic sink concentration can be achieved with relatively small movement of calcium from the two major stores
I.e. relatively little needs to be removed from the cytosol to get back to basal levels
Ca2+ concentration is much greater ________ the cell compared to _______ the cell
Outside
Inside
Cell membranes are normally ___________ to Ca2+ can only access the interior of the cell through highly selective _________/________
Impermeable
channels/transporters
Name the three major routes of calcium through the plasma membrane
Voltage-gated calcium channels (VGCCs)
Ligand-gated ion channels (LGICs)
Store-operated channels (SOCs)
Voltage-gated ion channels open in response to what?
Depolarisation
Most ligand-gated ion channels are activated by what kind of molecules?
Neurotransmitters
Store operated channels are distinctive Ca2+ channels with very low __________ that operate over hours, minutes or seconds?
Conductances
Seconds
Store operated channel conductance is relatively slow or fast compared to other channels?
Slow
Are SOCs expressed in excitable or non-excitable cells?
Both
In non-excitable cells, SOCs are important for what?
Accessing extracellular Ca2+ when SER stores for Ca2+ are depleted
SOCs are important in _______ muscle where prolonged states of stable _______ are required
smooth
contraction
The plasma membrane contains two very important carriers for Ca2+ that contribute to re-establishing the very low cytosolic basal Ca2+ concentration of _____.
What are they?
10^-7
Plasma Membrane Calcium ATPase (PMCA)
Sodium-Calcium Exchanger (NXC)
The PMCA is expressed in which cell types?
All cells
The PMCA uses how many molecules of ATP per ion of Ca2+ out of the cell?
One
The PMCA transfers Ca2+ from where to where?
From the cytosol to the extracellular space
Doe the PMCA have a high or low affinity for Ca2+?
High affinity
What is Calmodulin?
A calcium sensor
Is PMCA’s expression in cells normally quite high or low?
Low
PMCA’s general role is to “____________” intracellular Ca2+ concentration regulation
Fine-tune
What is NCX?
Sodium-Calcium exchanger
Does NCX require ATP?
No
How does the NCX work if it does not use ATP directly?
Uses the electrochemical energy gradient provided by the large concentration of extracellular Na+
The NCX exchanges _____ Na+ into the cell for every ______ Ca2+ pumped out
3
1
Following the work of the NCX, how are the intracellular and extracellular levels of Na+ restored?
The Sodium/Potassium (Na+/K+) ATPase
The NXC has a ______ affinity for Ca2+ than PMCA, but is expressed in ______ cell types.
Low
Many
Which NCX has a relatively ____ affinity for Ca2+, it has a _______ capacity for pumping Ca2+ out of the cell when required
Low
High
Compared to the PMCA, _____ is considered to act as the primary pump of Ca2+ when levels are over _______
NCX
10 microM
NCX is especially active in which tissues?
Excitable tissues such a muscle and nerve
What happens to the NCX in heavily depolarised cells?
The NCX is reversed and therefore pumps Ca2+ into the cell and pumps Na2+ out to rectify the increased concentration of intracellular Na+
Which three receptors are involved in controlling the release of Ca2+ from the SER/SR intracellular “store” into the intracellular cytosolic “sink”?
GPCR (Gq types) in the plasma membrane
IP3 receptors in the SER/SR membrane
Ryanodine (Ryr) receptors in the SER/SR membrane
What type of GPCR contributes to the regulation of Ca2+ efflux from intracellular stores?
Gq type GPCRs
In brief, explain what happens when an agonist binds to the Gq receptor type at the plasma membrane
-Phospholipase C (PLC) is activated by the alpha q subunit
-PLC cleaves PIP2 in the plasma membrane into IP3 and DAG
-IP3 then binds to IP3 receptors on the SER/SR membrane and activates them
-Leads to a large increase in intracellular Ca2+ released from the SER/SR
(-DAG activates PKC which activates its own substrates within the cell)
Why is it so important that intracellular levels of Ca2+ return to basal levels as soon as possible once they are raised?
Prolonged increased Ca2+ concentration in the cytoplasm can lead to cell death
What type of receptor is the IP3 receptor?
Ligand gated ion channel
What happens when IP3 binds to the IP3 receptor on the SER/SR membrane?
The IP3 receptor ion channel opens and allows Ca2+ efflux out of the SER/SR and into the cytosol
IP3 receptor activity remains _________ to the Ca2+ level driving the synthesis of IP3
Proportional
Which route is the main way in which GPCR activation signalling via increases in IP3 causing an increase in cytoplasmic Ca2+?
Binding of IP3 to IP3 receptors in the SER/SR membrane
What type of receptors are Ryanodine receptors (RyRs) ?
Ligand gated ion channels
IP3 and DAG are examples of what?
Second messangers
RyRs are expressed across cell types and are heavily expressed in the _____ of muscle cells
Sarcoplasmic reticulum (SR)
The activation of RyR channels varies in the three major muscle types which are…
Smooth, Skeletal, Cardiac
In smooth and cardiac muscle, the triggering calcium signal comes through the activation of which signalling molecule targets?
Voltage-operated Calcium Channels (VOCCs)
