When a ligand binds to GPCR it leads to a conformational change which activates the GPCR which activates a G protein which activates second messengers

Second messengers include cyclic nucleotides, phospholipids, and Ca^2+

Cyclic Nucleotides (cAMP)

For second messengers to work properly there must be a low basal level of it inside the cell.

-        G proteins activate adenylyl cyclase which catalyses the synthesis of cyclic AMP.

-        The upregulation is very short lived and cyclic AMP phosphodiesterase (PDE) brings the concentration down to basal levels

o   This is important as the transient upregulation means the cell can respond to a stimulus then a secondary stimulus

-        Gαs will increase cAMP, Gαi will increase cAMP

cAMP induces effects by activating cyclic-AMP dependant protein kinase (PKA)

-        Inactive PKA has a regulatory subunit which binds to cAMP to then dissociate to release the catalytic subunits

-        Activated PKA phosphorylates serine’s or threonine’s on selected target proteins, both intracellular signalling and effector proteins

Regulatory subunits of PKA

These are important for localising the kinase as they bind to both the catalytic subunit and A-kinase anchoring protein (AKAP)

-        AKAP binds to membranes of organelles, other signalling proteins or components localising the PKA and the plasma membrane

-        This creates a signalling complex changing the way cells can react

Basically:

-        AKAP binds both PKA and PDE. PDE hydrolyses cAMP

-        In unstimulated cells [cAMP] is lowered by PDE and in stimulated cells there is an increase in [cAMP]

-        [cAMP] is kept low due to this as when PKA is active it phosphorylates target proteins including PDE activating to bring the concentration of cAMP down which means there is only ever a short pulse of PKA activity

cAMP can have both long term and short term effects

Short term

Causes the phosphorylation of proteins in the cytosol

-        Such as opening a channel

Long Term

PKA moves into the nucleus and can alter gene transcription

-        PKA phosphorylates cAMP response element-binding protein (CREB) which binds to cyclic AMP response element (a sequence on DNA)

-        By binding to cAMP response element on the DNA, it activates CREB binding protein which binds to CREB activating the target gene causing gene transcription

Phospholipids

Many GPCRs exert their effects through Gαq proteins that activated plasma-membrane-bound enzyme phospholipase C-β (PLCβ).

-        When activated it cleaves PI 4,5-biphosphate (PI(4,5)P2) into two products

-        diacylglycerol (DIG) which activates protein kinase C

o   for protein kinase C to be activated it needs to be bound to DAG, phosphodiserum(?) and Ca++

o   Stays at plasma membrane

-        inositol 1,4,5-triphosphate (IP3) which causes the release of Ca++ from the endoplasmic or sarcoplasmic reticulum

o   moves into cytoplasm

Calcium Ion signalling

Ca++ functions as a major intracellular signalling mediator

-        In microsecond an increase in Ca++ in nerve cells causes exocytosis at synaptic endings

-        In milliseconds an increase in Ca++ can cause contraction in muscle cells

-        In minutes/hours Ca++ can drive processes like gene transcription and cell proliferation

Calcium ion signalling toolkit

Within the genome there are many genes that are the Ca++ signalling toolkit

-        Ca++ channels which increase concentration

-        Pumps and exchangers which decrease concentration

-        Buffers which bind to Ca++ and stop them from binding to sensors

-        Sensors to transduce the signal

As cells differentiate, they express a unique set of tool kit components causing unique signalling systems with different special and temporal properties which can respond in different ways and adapt to changing circumstances

Signalling

At basal levels when unstimulated there is a low concentration in the cytoplasm, high concentration in storage in the ER/SR and high concentration in extracellular fluid

-        Channels on plasma membrane or ER/SR to let Ca++ into the cytoplasm

-        This increase is detected by the cell and it will respond

Ca++ signal can be derived from internal or internal stores

-        External Ca++ enters the cell in response to:

o   Membrane depolarisation, stretch, noxious stimuli, extracellular agonist, intracellular messengers internal store depletion

o   There are a wide range of channels that mediate entry, links into the toolkit

§  Voltage-gated channels (VOCs)

§  Receptor-operated channels (ROCs)

§  Second-messenger operated channels (SMOCs)

§  Store-operated channels (SOCs)

§  Thermosensors

§  Stretch-activated channels

Ca++ Waves

-        Internal Ca++ enters the cytoplasm from the endoplasmic or sarcoplasmic reticulum is controlled by Ca++ or messengers such as:

o   Insositol-1,4,5-triphosphate (Ins(1,4,5)P3)

o   Cyclic ADP ribose (cADPR)

o   Nicotinic acid adenine dinucleotide phosphate (NAADP)

o   Sphingoine-1-phosphate (S1P)

-        IP3 gated Ca++ channel open, as the concentration increases ryanodine receptor (RYR) respond to rising Ca^2+ levels and amplify the Ca++ channel by opening IP3 gated channels on ER/SR which leads to a flood of Ca++. RYR receptors are inhibited by high concentration of Ca++ so they close

o   The positive and negative feedback produce Ca++ waves which the cell recognises and responds to

-        The waves are continuous as if the signal is still around IP3 is still being generated so it will cycle

The shape of the wave determines how the cell will respond to a wave, taking into account the duration, frequency and amplitude

Example

The fertilisation of an egg by a sperm triggers a wave of cytosolic Ca++

-        Sperm bring sperm-specific form of PLC which cleaves PIP2 to IP3

-        This releases Ca++ from the ER with IP3R casing an increase in Ca++

-        This wave changes the egg surface preventing the entry of other sperm while initiating embryonic development

Pumps and exchangers

Contribute to lowering the Ca++ concentration to basal levels

These include plasma membrane

-        Ca++ ATPase (PMCA)

-        Na+/Ca++ exchanger (NCX)

On the SR/ER there is

-        Sarco(endo)plasmic reticulum Ca++ATPase (SERCA)

On mitochondria there is the uniporter which temporarily transports Ca++ into the mitochondria

Sensors

There are two families of sensors, the C2 domain and EF-hand

When Ca++ is released the majority binds to buffers rather than sensors

Calmodulin

This is a major Ca+ sensor found in all eukaryotic cells making up to 1% of protein in a cell, highly conserved single chain with four high affinity Ca++ binding sites and belongs to the EF-hand family. Calmodulin cannot do anything without being bound to Ca++

-        Has EF-hand motif with a helix-loop-helix which bind to Ca++ causing a massive conformation change making it into a dumbbell shape

Role is to convert the ionic Ca^2++ signal into activation of intracellular signalling pathways

Ca++/calmodulin complex has no enzymatic activity itself, ats by binding to other proteins

-        An serve as a permanent regulatory subunit of an enzyme complex

-        The complex has many targets so calmodulin binding proteins are a very diverse group

CaM-kinases have 12 different subunits

-        A mixture of different subunits are activated dependant on the Ca++ wave

-        The different number of subunits that have become activated will catalyse different substrates

Exam Question

Discuss how components of the calcium-signalling toolkit influence the calcium signal, which in turn determines the cellular response

Channels, pumps, buffers, sensors

-        The different channels in the toolkit will determine what extracellular signals will cause the Ca++ channels to open, also the amount expressed by the cell can determine how quickly the cell can react to

-        Different types of pumps will determine the Ca++ wave

-        Different sensors will determine how the cell can use the Ca++