BIOMG 1350 Lecture 17 - Cell Signaling Part 2

Ion Channel Coupled Receptors

• Signal molecules cause a change of the permeability of the plasma membrane to specific ions, thus a change of membrane potential. Allows ions to flow in or out of the cell.

• They have very rapid responses (within milliseconds) and are especially important in nerve cells and muscle cells.

• Neuronal signaling uses ion channel coupled receptors. When a signal bind to the ion channel coupled receptor, it opens the channel allowing sodium ions to flood into the post synaptic cell, allowing the cell to be activated and to perform its function.

• The post synaptic cell is the cell that receives the neurotransmitter from the activated nerve terminal from the signaling neuron. The neurotransmitter signaling ligand crosses the synaptic cleft to reach the inactive neurotransmitter receptor (transmitter gated ion channel) to cause a change in membrane potential (electrical signal).

GPCR Pathway at a Glance

• G protein coupled receptor (GPCR) pathways at a glance. The process is how the GPCR pathway goes from signal to response.

  • Signal (hormones, local mediators, neurotransmitters, light, etc.).

  • GPCR (g protein couple receptor, humans have around 800 different kinds of these).

  • G Protein (20 different G proteins and each is activated by a specific set of receptors and each activates a particular set of downstream targets).

  • Targets (ion channels or membrane bound enzymes. Most frequent targets are the enzymes: Adenylyl cyclase -> cAMP and Phospholipase C -> Ca2+/DAG).

  • Responses (many possible responses of which include secretion, contraction, heart rate, glycogen or fat breakdown, smell, taste, vision, etc.)

GPCRs

All GPCRs have a similar structure and they are the largest class of receptors. They are seven pass transmembrane proteins that can bind to a ligand from the extracellular space.

Cell-surface receptors that detect extracellular signaling molecules (ligands) and activate intracellular G proteins to initiate signal transduction pathways. They trigger cellular responses by promoting the exchange of GDP for GTP on G proteins.

Both GPCRs and G proteins have an ON and OFF state.

G Proteins

• They are different from the monomeric small G proteins (Ras, Ran, Rab, etc.). Even though G proteins have GDP/GTP exchange for activation, they are not the same as the previous GTP/GDP proteins we have spoken about previously is basically what this is saying.

• G proteins have three subunits (trimeric): alpha, beta, and gamma.

• Intracellular signaling proteins that act as molecular switches, transmitting signals from activated receptors to target proteins inside the cell. They are active when bound to GTP and inactive when bound to GDP, helping regulate cellular responses.

• Both GPCRs and G proteins have an ON and OFF state.

GPCR Activation of G Proteins

• Signal binding in GPCR by a ligand causes a conformational change in the receptor that is transmitted to its cytoplasmic domain. This interacts with the G protein on the intracellular side of the membrane. An activated receptor acts as a GEF for its G protein basically. It exchanges GDP for GTP in the G protein.

• Once the G protein is activated by GPCR, the activated G alpha and G gamma and beta separate and each of these two subunits can activate different effector proteins.

• Once activated, a single GPCR can activate many molecules of a G protein.

GPCR Inactivation

• GPCR inactivation occurs through two major processes.

  • A GPCR is deactivated when a signal molecule is no longer available.

  • Even if a signal remains bound to a receptor, a GPCR is eventually deactivated and recycled (the whole protein).

G Protein Inactivation

• G Protein inactivation occurs via hydrolysis of GTP.

  • A G protein is inactive when the G alpha subunit hydrolyzes GTP to GDP after it finishes its function.

  • The inactive G alpha subunit then reassembles with the G beta and gamma subunits to reform the G protein waiting for another single from a GPCR.

How G Proteins Work to Transduce the Signal Downstream

• Many G proteins activate membrane bound enzymes that produce small messenger molecules. Specifically, the G alpha subunit of the G protein activates enzymes related to production of small second messenger molecules.

• Two main targets of GPCRs or specifically the activated G protein alphas subunit are adenylyl cyclase and phospholipase C (PLC) which are transmembrane enzymes. These among other second messenger molecules diffuse to act on intracellular signaling proteins.

GPCR and adenylyl cyclase activation process (example of G protein signal transduction)

• First the signal (epinephrine, in this case) binds to the GPCR. Then, this activates the alpha subunit of the G protein (can sometimes be the G protein beta or gamma that is activated), which in turn activates the transmembrane enzyme adenylyl cyclase.

• The transmembrane adenylyl cyclase cleaves ATP to make cyclic AMP (cAMP), which is a second messenger.

• cAMP activates protein kinase A (PKA), which phosphorylates effector proteins, including transcription regulators.

Examples of GPCR activation of Adenylyl Cyclase

• Some cell responses mediated by cAMP include: Epinephrine to the heart to increase heart rate and force of contraction, Epinephrine to skeletal muscle to induce glycogen breakdown, epinephrine or glucagon to fat to cause fat breakdown, and then ACTH to the adrenal gland to cause cortisol secretion.

• One specific example is the cholera toxin. The cholera toxin prevents the hydrolysis of GTP to GDP so the G protein alpha subunit is constantly active (once the GPCR is activated).

• This Leads to the continuous production of cAMP, continuous activation of PKA, and phosphorylation of effectors.

• These effectors include channels that allow ions and water to flow out of intestinal epithelial cells into the intestinal lumen, causing diarrhea.

GPCR and phospholipase C (PLC) activation process (example of G protein signal transduction).

• First the signal molecules binds to GPCR. Then, this activates the G protein alpha subunit (can sometimes be the G protein beta or gamma that is activated).

• The G protein alpha subunit activates phospholipase C, which cleaves the inositol phospholipid PIP₂ into IP₃ and DAG. These two second messengers then act through different signaling pathways.

• Phospholipase C leads to the production of two second messengers. These second messengers are diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3).

• IP3 can lead to the release of Ca2+ from the ER, thus increase cytosolic Ca2+ levels. Ca2+ acts as a second messenger to trigger many biological processes. Thus IP3 second messenger binds to a Ca2+ release channel on the ER to release more Ca2+ second messenger.

• DAG, in combination with Ca2+, can activate protein kinase C (PKC). Protein Kinase C phosphorylates diverse effector proteins and is calcium dependent. Thus PKC requires Ca2+ from IP3 second messenger and also requires DAG.

Examples of GPCR activation of phospholipase C

Some cell responses mediated by phospholipase C activation:  vasopressin targets the liver and causes glycogen breakdown, acetylcholine targets the pancreas and causes a secretion of amylase, acetylcholine also targets smooth muscle to cause contraction, and thrombin targets blood platelets to cause aggregation.

Wider Scientific and Medical Applications of GPCRs, G proteins, and GPCR Specific Pathways

• The discovery of G proteins, GPCRs, and specific GPCR pathways have led to multiple Nobel prizes. (1994 discovery of G proteins, 2004 discovery of odorant receptors and organization of the olfactory system, and 2012 discovery of GPCRs).

• The pharmaceutical industry invests heavily in these pathways. About a third (38.4% of drugs work through GPCRs).

Enzyme Coupled Receptors

• Receptors can act as enzymes or receptors can associate with enzymes. There is no middle step like the prior examples between receptor and enzyme. The receptor either is or directly interacts with the enzyme.

• Signal molecules in the form of a dimer can bind to inactive catalytic domains to activate the catalytic domain. This occurs in certain enzyme coupled receptors such as RTK.

• Some receptors can simply bind to a ligand and undergo a conformational change that activates signaling, without needing to dimerize.

• One example is Receptor Tyrosine Kinases (RTKs) (Type of enzyme coupled receptor that acts as an enzyme itself)

Receptor Tyrosine Kinases (RTKs)

• (Type of enzyme coupled receptor that acts as an enzyme itself)

  • Receptor tyrosine kinases (RTKs) represent one of the largest classes of enzyme coupled receptors.

  • They are activated by growth factors (signals), such as FGF (fibroblast growth factor), EGF (epidermal growth factor), etc.

  • Their cytoplasmic domains function as tyrosine protein kinases. They regulate growth, proliferation, differentiation and survival of cells.

  • RTKs can dimerize and one of the kinase domains can phosphorylate the other in a process called autophosphorylation to allow the RTK as a whole to activate intracellular signaling proteins.

Receptor Tyrosine Kinase (RTKs) Function

• A signal molecule in the former of a dimer can bind to the RTK extracellular domains that are apart, bringing both together and activating the kinase activity.

• Ligand doesn't have to be a dimer but in many cases a single ligand molecule binds two receptors and pulls them together.

• Once kinase domains are activated by the ligand, they phosphorylate each other in a process called autophosphorylation. The resulting phosphotyrosines serve as docking sites that recruit intracellular signaling proteins and activate them, which in turn activates downstream intracellular signaling pathways.

Receptor Tyrosine Kinase (RTKs) and Ras

• Most RTKs activate the small GTPase Ras. The activated intracellular signaling proteins from the phosphorylated RTK can activate Ras GEF which activates Ras GTPase by swapping the GDP for GTP to transmit a signal.

• This signal can act on its own intracellular signaling pathway eventually toward an effector that performs some function.

• Ras activation can lead to cell proliferation and around 30% of cancers have activating mutations in Ras. This is because Ras is involved in cell proliferation.

Interconnectedness of Signaling Pathways

• All 3 major classes of cell surface receptors (ion channel coupled receptors, g protein couple receptors, enzyme coupled receptors) work together.

• For example, a GPCR can activate a pathway which is also influenced (perhaps through signal integration) by a RTK. These both can lead to different forms of altered cell behavior.

• Animals integrate and distribute multiple signals to produce diverse responses and the 3 major classes of cell surface receptors can produce intracellular signaling pathways are the starting points that allow this to happen. This also proves how signaling pathways are interconnected.

• Signals can be integrated and distributed to cause everything from apoptosis to survival growth and division or differentiation.

Sea Urchin Polyspermy During Fertilization Studies

• Sea Urchins are a great model system for studying fertilization as they are readily available and can be kept in a laboratory, have external fertilization, make many gametes, and can be induced to release gametes upon injection with KCl.

• The biological problem: Fertilization occurs when one sperm fuses with an egg. How does the egg ensure that only one sperm fuses, when it is surrounded by dozens or even hundreds of sperm.

• Sometimes for sea urchins, multiple sperm cells can fertilize one egg. This results in many consequences.

Consequences of polyspermy (in Sea Urchins)

• Adult sea urchins are diploid (18 pairs of chromosomes or 36 in total) and their gametes (egg and sperm) are haploid (18 chromosomes each). The sperm also contributes 2 centrosomes.

• A normal fertilized egg has 36 chromosomes and 2 centrosomes. A egg fertilized by 2 sperm contains 54 chromosomes and 4 centrosomes.

• A normal fertilized egg metaphase and anaphase looks quite typical with two daughter cells forming, but an abnormal egg fertilized by two sperm has 4 centrosomes and chromosomes pulled apart in 4 directions (NSEW) with 4 daughter cells forming.

• Polyspermy leads to disintegrating cells and the death of the embryo as the 4 daughter cells (due to the 54 chromosomes and 4 centrosomes from 2 sperm entering one egg) each contain a different and not sufficient number of chromosomes.

Fast Block to Polyspermy

• The fast block to polyspermy involves an egg that contains a positive membrane potential as the sperm cannot fuse with an egg that has a positive membrane potential.

• What type of cell surface receptor is used for the fast block? An ion channel coupled receptor as it is the fastest.

• Basically the egg resting cytosolic membrane potential is -70 mV and sperm can bind and fuse with the egg. The moment a sperm fuses with the egg, ion channels open letting Na+ inside the egg causing its membrane potential to go from -70 mV to +20 mV preventing any other sperm from entering.

• However, the fast block to polyspermy in sea urchins isn't sufficient as after 60-90 seconds, the membrane potential slips back down below 0 (negative) which could allow another sperm to fuse. Thus another, slow block, is needed to permanently prevent sperm from entering the egg.

Slow Block to Polyspermy

• Cell signaling in response to sperm binding causes the release of Ca2+ from the egg's ER, triggering the slow block. The outer layer of the egg lifts away from the surface preventing any more sperm from fusing (basically a fertilization envelope or shield is created).

• Under a fluorescent microscope, a calcium wave can be seen to spread through the egg after the first sperm enters which causes this slow block.

• This slow block to polyspermy uses the phospholipase C pathway. The sperm binds to a receptor and activates a tyrosine kinase which activates a phospholipase C which sends IP3 to bind to a C2+ channel on the ER to let Ca2+ escape into the cytosol.

• This escaped Ca2+ initiates changes to the outer layer of the egg (slow block).

• The activated phospholipase C also activates DAG which then, with the help of Ca2+ released from IP3 binding, activates PKC which activates the egg from its previous dormant state.

Ca2+ Role in Preventing Polyspermy

• Ca2+ sweeps across the egg when released from the ER in 30-60 seconds. It acts as a second messenger in the slow block to polyspermy which activates proteins that cause the outermost layer of the egg to lift off the surface, forming a protective fertilization envelope that prevents sperm from binding to the egg.

• Ca2+ is released when the IP3 from the phospholipase C activity binds to an ion channel on the outer part of the ER membrane releasing Ca2+.

• Ca2+ can also combine with DAG to activate protein kinase C (PKC) which in the context of development, brings the maternal egg out of its dormant state.

Prevention of Polyspermy Overview

• Fusion of egg and sperm leads to the activation of two things.

• Na+ Influx from an ion channel coupled receptor which leads to membrane potential change which is the fast block to polyspermy (as sperm cannot fuse with egg if it has positive membrane potential).

• Kinase stimulation which results in phospholipase C activation. This activates IP3 which releases Ca2+ from the ER which in turn causes changes to the outer shell of the egg to initiate the slow block.

• The Ca2+ released also activates Protein Kinase C along with activated DAG from the Phospholipase C and PKC causes egg activation (stimulation of protein synthesis, DNA replication, cytoplasmic movements of morphogenic materials).