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1

What is Signal Transduction?

Majority of extracellular signalling molecules do not readily cross the plasma membrane and therefore their eceptors are located at the cell surface.

Althogh some receptors can directly alter cellular activity, many require "transduction" of the initial ligand binding event via other intraelular signalling components to generate a response e.g. proliferation, secretion

2

What are the 3 superfamilies of cell-surface receptors?

  •  Ligand-gated (receptor-operated) ion channels such as nAChR (on the membrane of skeletal muscle cells)
  • Receptors with intrinsic enzyme activity (receptor tyrosine kinases e.g. insulin receptor)
  • G-Protein Coupled (7TM) Receptors e.g. mAChR

 

Each receptor subtype is specific for one (or a very lmited number of) chemical endogenous (ligand)

3

How do Receptors with intrinsic enzymatic activity work?

  • Ligand binding activates an enzyme activity e.g. tyrosine kinase.
  • Tyrosine kinase phoshorylates the receptor itself and other substrates.
  • The beta-transmembrane spanning domains contain tyrosine residues within them. Once the Tyrosine reisudes are phosphorylated, this leads to a signalling cascade, attract other molecules.

4

What cellular functions are the GPCRs responsible for?

Wide diversity including:

  • Muscle contraction
  • Stimulus-secretion coupling
  • Catabolic and anabolic metabolic processes
  • Light, smell and taste perception

 

GPCRs alter the activities of effectors which may be second messenger-generating enzymes (e.g. adenyly cyclase) or ion channels, via activation of one or more types of guanine nucleotide binding protein (G proteins)

5

Give some examples for the usage of therapeutics targeting GPCRs and some diseases associated with signal transduction

  • Currently ~40% of all available presciption drugs exert their therapeutic effects directly (as agonists or antagonists) or indirectly at GPCRs.
  • Agonists bind to te receptor and actvate it (leading to intracellular signal transduction events) e.g. anti-asthma, (Salbutamol and Salmeterol which are B2 agonists and cause brachiodilation, facilitate oxygen exchange, relaxation of smooth muscle) and analgesia/anaesthesia (morphine and fentanyl which are m-opoid receptor agonists - pain relief)
  • Antagonists bind to the receptor but do not activate it e.g. Hypertension (propranolol and atenolol are B-adrenoceptor antagonists)

6

What happens when there are mutations to GPCRs?

  • Occurs in rare diseases
  • Results in loss-of-funcion or gain-of-function e.g.
  • Retinitis pigmentosa can be caused by  loss-of-function mutation to rhodopsin.
  • Nephrogenic diabetes insipids can be caused by a loss of function mutation to the V2 vasopressin receptor.
  • Familar male pecocious puberty is caused by a gain of function mutation to the luteinizing hormone (LH) receptor. The receptor is continuously active even in the absence of a ligand.

7

What types of stimuli do different GPCRs respond to?

  • Ions (H+. C2+)
  • Neurotransmitters e.g. ACh, glutamate
  • Peptide and non-peptide hormones e.g. glucagon, adrenaline
  • Large glycoproteins e.g. Thyroid-stimulating Hormone

8

Describe the structure of GPCRs

  • single polypeptide chain (300-1200 amino acids)
    7-transmebrane spanning reions
  • Extracellular N-terminal
  • Intracellular C-terminal

9

What regions of the GPCR are responsible for ligand binding?

  • For some reeptors the ligand binding site is formed 2-3 transmembrane domains; the ligad binding site is hidden deep down between transmembrane domains; generally small ligands such as ACh and NA bind in the core between transmembrane domains.
  • In others, the N-terminal region (and other extracellular domains) form the ligand binding site

10

What does an activated GPCR interact with?

  • A guanine nucleotide binding protein (G-Protein)
  • G proteins are heterotrimeric; made up of 3 subunits (alpha, beta and gamma) but they act as a single functional receptor.
  • The G protein alpha-subunit has a guanine nucleotide binding site which bind GTP and sowly hydrolyses it to GDP (i.e. the alpha-subunit possesses GTPase activity).
  • Under basal conditions the G protein is present at the inner face of the plasma membrane membrane predominantly in its heterotrimeric form with GDP bound to the alpha-subunit.

11

What happens after the GPCR has been activated by ligand binding?

The activated receptor undergoes a conformational change causing activation of G-protein.

  • The GPCR has a high affinity for the normal basal condition form of G-Protein and a protein-protein interaction occurs leading to GDP being released and GTP binding in its place onto the alpha-subunit; the GPCR act as a guanine nucleotide exchange factor (GEF). This exchange activates the G-Protein
  • The binding of GTP decreases the affinity of alpha-GTP for the receptor and for the G-beta-gamma subunit.
  • Thus alpla-betagamma immediately dissociate into alpha-GTP + free beta-gamma subunits and each can then interact with effector proteins.
  • All of this takes place on the inside of the membrane

12

How long does the a-GTP and/or By interation with effectos last?

  • Until the effector interaction is terminated by the intrinsic GTPase activity of the G-alpha-subunit
  • When this occurs the affinity of the alpha-subunit for a beta-gamma subunit increases and the G-protein heterotrimer is reformed and awaits reactivation by an agonist-activated receptor to re-initiate the cyle.

13

Why can the G-protein be thought of as an on/off switch and a timer?

  • The on switch is receptor-facilitated GDP/GTP exchange
  • The timer/off switch is governed by the length of time taken for GTP hydrolysis on the G-alpha-subunit
  • There is increasing evidence that the timer function may not be a fixed property of the G-alpha subunit but may also be regulated by other cellular proteins eg. RGS proteins

14

Give some examples of G-Protein Diversity

  • There are >1000 possibe GaB-y combinations
  • Activated GPCRS preferentially interact with specific types of G protein.
  • The alpha subnit is a primary determinant.
  • In turn G-alpha and beta-gamma subunits interact with specific effector proteins.
  • In this way, an extracellular signal, working via specific GPCR, will activate a single or small-sub-population of G proteins and effectors in the cell to bring about a specific cellular response

15

Describe the interactin of G(q) proteins

 Preferentially interact wth membrane bound enzyme phospholipase C causing hydrolysis of a minor plasma membrane phospholid, PIP2, to generate 2 second messengers - IP3 and DAG

16

What kind of G protein does rhodopsin activate?

The light sensing protein rhodopsin, present in mammalian retinal rod cells, activates a G protein called Transducin or G(t), which activates a phosphodiesterase enzyme which hydrolyses the second messenger cGMP to 5'GMP.

17

Describe how Cholera toxin (CTx) and Pertussis toxin (PTx) interfere with G Protein function

  • CTx and PTx are enzymes that ADP-ribosylate specific G proteins
  • CTx elimiates GTPase activity of G(s)-alpha, leadig to G(s)-alpha becoming irreverisbly activated
  • PTx interferes with GDP/GTP exchang on G(i)-alpha, leading to G(i)-alpha becoming irreversibly inactivated,.

18

Link receptors to the G-alpha subunit types they utilise

19

What kind of effectors could GPCRs lead to the activation of?

GPCR --> G Protein --> Effector

The G Protein is a peripheral membrane associated protein.

Effectors can be ENZYMEs: Adenylyl Cylase, Phosholipase C, Phosphoinsitide 3-Kinase (PI3K), cGMP Phosphodiesterase (present in retinal rod and cone cells)

Or Effectors can be Ion Channels; Voltage-Operated Ca2+ Channels or G-Protein-Regulated Inwardly-Rectifying K+ Channels (GIRKs)

NOTE: GIRKs are important in the heart, M2 receptors present in SAN regulated by GPCRs, coupled to G(i) proteins, activated by ACh

20

Describe Agonist-Stimulted Regulation of Adenylyl Cylase

  • Adenylyl Cylase is an integral plasma membrane protein which can be either activated (via G(s)) or inhibited (via G(i)) by activation of diferent eceptors.
  • Adenylyl Cylase hydrolyses cellular ATP to generate cyclic AMP
  • cAMP interacts with specific protein kinase (cAMP-dependent protein kinase, or PKA)
  • PKA can in turn phosphorylate a variety of other proteins within the cell to affect their activities.
  • cAMP could interact with other proteins such as Epacs (for small GTPases)and cyclic-nucleotide-gated ion chanels (CNGs) but cAMP exerts the majority of its actions through PKA.

21

What happens during resting conditons (regarding the agonis-stimulated regulation of adenylyl cylase pathway)?

  • During resting conditions, R (Regulatory) subunits sit in the cataytic sites inactivating the C (Catalytic subunits).
  • When there is increased [cAMP], R subunits become saturated with cAMP --> C subunits are released free into the cytoplasm.
  • C subunits act as protein kinases using ATP and phosphorylate proteins (serine and threonine) residues. They are specific, only phosphorylate key proteins at key sites to regulate their activity.
  • Variety of neurotransmitters use this pathway : NA, dopamine, histamine 
  • In this way receptors which activate adenylyl cyclase and incease cAMP can cause increased glycogenolysis and gluconeogenesis in the liver, increased lipolysis in adipose tissue, relaxation of a variety of types of smooth muscle and positive inotropic and chronotropic effects in the heart.

22

Describe the Phospholipase C signalling pathway

  • Phospholipase C hydrolyses a minor plasma membrane phospholipid, phosphatidylinositol 4,5-biphosphate (IP2) to generate 2 second messenagrs - InsP3 (Inositol 1,4,5-triphosphate) and DAG (diacylglycerol)
  • InsP3 exerts its effects by interacting with specific intracellular receptors on the endoplasmic reticulum to allow Ca2+ to leave the lumen of the ER and enter the cytoplasm.
  • One Consequence is the activation of Ca2+ sensitive protein kinases.
  • DAG activates a family - Protein Kinase Cs
  • A large number of GPCRs can activate this pathway and phospholipase C activation is mediated by G(q) proteins.
  • Thi signallin pathway is responsible for an array of important responses including vascular, GI tract and airways, smooth musle contraction, mast cell degranulation and platelet aggregation.
  • NOTE: IP3 receptor is a ligand-gated ion channel.

23

Describe the Cyclic GMP Phosphodiesterase Activity

  • Specialized mechanism found in the photoreceptive cells of th retina (Rod and cones).
  • in this case the breakdown of a second mesenger, cGMP, is regulated by activation of cGMP phosphodiesterase by G(t) (Transducin) following excitation of rhodopsin by a photon of light.
  • In the dark, levels of cGMP are sufficient to open a second messenger-operated on channel which allows Ca2+ and Na+ to enter the cytoplasm.
  • On exposure to light, activation of cGMP phosphodiesterase causes a decrease in cGMP leading to channel closure and membrane hyperpolarization, thus altering the signal output to the CNS

24

What do increases in cAMP, cGMP, DAG and Ca2+ all have in common?

They exert all or at least a part, of their actions via interactions with specific serine/threonine protein kinases (so called, because these kinases phosphorylate specific serine and/or threonine amino acid residues within the target proteins).

Each protein kinase causes phosphorylation of a distinct family of target proteins whose activities are increased, decreased or unaltered by this covalent modification

25

Discuss why amplification is necessary and how it can be achieved.

  • For an extracellular stimulus which may only be a few molecules of a hormone interacting with appropriate cell surface receptors, to generate an intracellular response, amplification of the sigal is essential.
  • This can be achieved in a number of ways.
  1. Actvated receptor can cause (sequential) GTP/DP exchange on more than one G-Protein
  2. An activated a-GTP subunit/ free By-subunit can activate multiple effector molecules.
  3. Effector molecules act catalytically. Thus activation of adenylyl cyclase results in conversion of 100-1000 of ions to move across the plasma membrane.

26

How is further amplification often achieved?

Further amplication is often achieved through the mechanisms by which the second messengers activate their cellular targets as these often involve an enzyme e.g. cyclic-AMP dependent protein kinase, protein kinase C) or a sequence of enzymes (i.e. an enzyme cascade).

 

 

27

Both activation AND de-activation of signalling pathways is rapid, generally occurring over a time-scale of a few seconds. Deactivation is facilitated by a number of aspects of signalling pathways - which are?

 1. Once a receptor has productively interacted with a G protein the binding of agonist molecule is weakened and agonist-receptor dissociation is more likely to occur.

2. Whilst activated the receptor is susceptible to a variety of protein kinases which phosphorylate the receptor and prevent it activating further G proteins (this is an important part of the receptor densensitization phenomenon observed, for most, but not all, GPCRs)

3. The active lifetime of α-GTP may be limited by cellular factors which stimulate the intrinsic GTPase activity of the Gα-subunit.

4. Enzymatic activities in the cell are such that the basal state is favoured. Thus, cells contain high activities of enzymes which metabolize second messengers (e.g. cyclic AMP is metabolized to the non-biologically active 5’-AMP by phosphodiesterases)

5. Similarly enzymic cascades activated downstream of second messenger/ protein kinase activation are opposed by activities which act to reverse the second messenger/protein kinase effect (i.e. target protein phosphorylation is reversed by active cellular protein phosphatase activities)

28

Describe the regulaton of Chronotropy in the heart signalling pathway

The intrinsic rate at which the SA node fires an action potential can be affected by ACh released by the parasympathetic nerves. The predominant ACh receptor population in the SA node is M2-muscarinic cholinoceptors; activation of these receptors increases the open probability of K+ channels which have been shown to be directly regulated by both αi-GTP (and perhaps the βγ-subunits simultaneously released).

Although M2-muscarinic cholinoceptor activation will also inhibit adenylyl cyclase activity, it is not known whether this has any functional consequences.

The increased plasma membrane K+-permeability causes a hyperpolarization which slows (and if strong enough prevents) the intrinsic firing rate resulting in a negative chronotropic effect.

29

Describe the regulation of Inotropy in the Heart signalling pathway

Sympathetic innervations of the cardiac ventricles (and/or circulating adrenaline) can influence the force of contraction (inotropy).

Activation of β-adrenoceptors (predominantly β1-Adrenoceptors) increases both cyclic AMP formation and the open probability of voltage-operated Ca2+-channels (VOCCs).

The increase in Ca2+ influx is brought about by two complementary mechanisms: β1-Adrenoceptors activate adenylyl cyclase via αs-GTP and the increase in cyclic AMP activates cyclic AMP-dependent protein kinase which can phosphorylate and activate the VOCC. In addition, αs-GTP can interact directly with VOCCs.

Thus the direct and indirect actions at the level of VOCCs reinforce each other and cause an increase in the magnitude of Ca2+-entry resulting in positive inotropic effect

30

Describe the Arteriolar Vasoconstiction Signalling Pathway

Release of noradrenaline acts on α1-adrenoceptors to stimulate phospholipase C and phosphoinositide turnover via a Gq protein.

The immediate effect is the generation of InsP3 which releases ER Ca2+ and initiates a contractile response. The role of DAG is less clear, but it is thought that activation of protein kinase C by DAG and the phosphorylation of key target proteins are important for sustaining the vasoconstrictor response to noradrenaline.