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Cellular Physiology > Cellular Signalling (Dr. Chidiac 18-23) > Flashcards

Cellular Signalling (Dr. Chidiac 18-23) Flashcards

1
Q

Generally, how do cells communicate?

A

Cells communicate and coordinate activities by sending and receiving signals. Each cell expresses a variety of receptor proteins that allow it to respond to particular signals. Signals originate from other cells and the environment.

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2
Q

What are the types of environmental signals?

A
  • Sensory signalling: detection of external signals by sensory receptors for light (rhodopsin), smell (olfactory, GPCRs), taste (GPCRs, ion channels, transporters), sound, balance, touch (mechanoreceptors)
  • Cellular environment: cells detect nutrients, protons, nucleic acids, osmotic pressure, fluid sheer stress, and xenobiotics (drugs outside the body + toxins)
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3
Q

Describe the different endogenous extracellular signalling molecules

A
  • Secreted from signalling cell into the extracellular space e.g. hormones, neurotransmitters
  • Released by passive diffusion through channels in PM e.g. ATP, prostaglandins
  • Liberation of PM-embedded ligands by matrix metalloproteinases (protealyse) e.g. growth factors, cytokines
  • Exposed to the extracellular space, but remain tightly bound to the surface of the signalling cell (protrude the surface of the cell) e.g. ephrins
  • Extracellular matrix proteins e.g. integrins
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4
Q

What are the different modes of intercellular signalling?

A
  • Endocrine signalling
  • Paracrine signalling
  • Synaptic signalling
  • Contact-dependent signalling / juxtacrine signalling
  • Autocrine signalling
  • Protease-dependent signalling
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5
Q

What is Endocrine signalling?

A
  • intercellular signalling
  • hormone signalling
  • endocrine cells release hormones into the bloodstream or lymphatic. system - transported throughout body (signalling over long distances)
  • hormones act on target cells that express a receptor for the ligand
    examples:
  • insulin released from the pancreas promotes glucose uptake throughout the body
  • parathyroid hormone (PTH) is secreted from the parathyroid gland and acts on PTH receptors in bone, kidney and intestine
  • hormone signalling is relatively slow compared to other modes (long distance)
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6
Q

What is Paracrine signalling?

A
  • secreted signalling molecules act as local mediators that impact target cells in the immediate environment
  • para = near
    examples:
  • platelet-derived growth factor (PDGE), wound healing
  • locally released parathyroid hormone related peptide (PTHRP) activates PTH1 receptors in bone and regulates mineralization
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7
Q

What is synaptic signalling?

A
  • specialized form of paracrine signalling
  • regulates neuronal communication
  • allows for adaptive changes required for behavioural responses or reflexes
  • neurons (nerve cells) extend long axons
  • axons contact each other at synapses
  • neurons can also form synapses with muscle cells
  • rapid mode of signalling
  • neurotransmitters include acetylcholine catecholamines (dopamine, adrenaline, noradrenaline), serotonin, endorphins, GABA, endocannabinoids, corticotropin releasing factors, etc.
  • typically stored in intracellular vesicles and released into synapses
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8
Q

What is contact-dependent signalling aka juxtacrine signalling?

A
  • physical contact between adjacent cells (or between cell and ECM)
  • “ligand” protruding from one cell activates receptor in plasma membrane of neighbouring cell (ephrin receptors, adhesion GPCRs)
  • Signalling molecules remain bound to surface of signalling cell
  • Activate target cells that come into contact with signalling cell
  • Important in developmental biology
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9
Q

What is Autocrine signalling?

A
  • cell releases a signalling molecule that acts back upon receptors expressed on its own surface to control its activity
  • auto = self
    examples:
  • cytokine interleukin-1 in immune cells
  • vascular endothelial growth factor (VEGF) in cancer cells
  • neurotransmitters (e.g. noradrenaline, 5HT) which bind to presynaptic autoreceptors (receptors measure how much neurotransmitter present)
  • positive/ negative feedback
  • apoptosis
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10
Q

What is protease-dependent signalling?

A
  • proteolytic activation of precursors
    examples:
  • proteinase-activated receptors: tethered ligand on GPCR amino-terminus is exposed by proteolysis
  • release of membrane-anchored ligand from extracellular surface by metalloproteinase activity
  • conversion of pro-hormone to active form by proteinase activity intracellularly (e.g. endorphins) or extracellularly (e.g. angiotensin II)
  • protease cleaves ligand, allowing activation angiotensin I. to angiotensin II
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11
Q

From the perspective of the receiving cell, what’s the difference between hormonal, paracrine, and autocrine signalling?

A
  • doesn’t really know - just reacts to the things in the environment, same machinery reacts to all of these methods
  • for endocrine may have higher affinity because hormones coming from further distance
  • some endogenous ligands can play multiple signalling roles, e.g. noradrenaline can act as a hormone, a neurotransmitter or an autocrine factor
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12
Q

How do cells recognize and respond to signals in their environment?

A

Cell surface receptors transduce signals into cells. Cell takes info and converts into change in its behaviour. Allows to react to environment

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13
Q

What does transduce mean?

A

To convert (something, such as energy or a message) into another form. Signal transduction.

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14
Q

What does affinity mean?

A

How tightly a drug binds to its receptor (KD= koff/kon), smaller KD = higher affinity, how fast/ slow something goes in or comes out from the binding site.

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15
Q

What does specificity/ selectivity mean?

A

A receptor binds preferentially to ligands that fit well into its binding site

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16
Q

What does saturability mean?

A

Ligand binding is limited by the number of receptors

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17
Q

What does reversibility mean?

A

The ability to bind to and dissociate from a receptor. Most ligands bind reversibly.

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18
Q

What does competition mean?

A

A binding site can only accommodate one ligand at a time. If two or more ligands are present they will compete with each other.

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19
Q

What does agonist mean?

A

An endogenous ligand or drug that binds to a receptor and promotes activation

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20
Q

What does antagonist mean?

A

A ligand that binds to a receptor but does not activate it.

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21
Q

What are the cellular determinants of receptor signalling?

A
  1. Receptor expression level:
    - availability of receptors will determine cellular response
    - cells that lack receptors for a particular signal cannot respond
    - cells with greater receptors density may respond at lower agonist concentrations and/or activate minor pathways
  2. Receptor variants
    - ligand may binding to distinct receptor subtypes in different cells
  3. Intracellular signalling components
    - same receptor may activate different pathways in different cells
    - depends on intracellular factors available to integrate and interpret receptor signal
    - e.g. complement of kinases, ion channels, phosphatases, substrates
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22
Q

How are the signals turned off?

A
  • endogenous agonists may be deactivated metabolically: hydrolysis of acetylcholine by cholinesterases, and breakdown of peptide and protein hormones by proteases
  • endogenous agonists may be reabsorbed from extracellular space via specialized uptake proteins (e.g. transporters for dopamine, adrenaline, noradrenaline, serotonin, prostaglandins)
  • drugs that mimic endogenous agonists can be metabolized by liver enzymes and/or excreted e.g. via the urine
  • receptor activation can also trigger intracellular processes that limit signalling, such as G protein uncoupling and receptor internalization
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23
Q

What are two types of molecular switches?

A
  • GTP binding and hydrolysis
  • protein phosphorylation/ dephosphorylation
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24
Q

Describe GTP binding and hydrolysis as a molecular switch. What are the regulation proteins of this?

A

Heterotrimeric G proteins and small Ras-like G proteins belong to a superfamily of GTPases where activation and deactivation correspond to GTP binding and hydrolysis, respectively.
For most G proteins, these changes occur on a time scale of seconds to minutes (i.e. slow relative to many biochemical processes)
Regulation of G protein activation state by other proteins:
- GEFs (guanine nucleotide exchange factors) promote GDPs dissociation and thus facilitate activation by GTP
- GAPs (GTPase accelerating proteins) promote the hydrolysis of GTP, thereby deactivating G proteins
- GDIs (guanine nucleotide dissociation inhibitors) inhibit GDP dissociation and thus impede activation

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25
Q

Describe protein phosphorylation/ dephosphorylation as a molecular switch.

A

Protein phosphorylation:
- ubiquitous strategy
- reversible covalent modification, with proteins dephosphorylated by phosphatases
- kinases undergo conformational changes in response to diverse inputs, which then regulate kinase catalytic function (phosphorylation)
- ATP cleaved to ADP; phosphate released covalently attached to a protein
- phosphorylation does not always mean activation
- often used downstream after initial binding - secondary messengers - often increase activation

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26
Q

What are the general characteristics of cell signalling?

A
  • endogenous agonist or mimic 1st messengers
  • receptors bind agonists and regulate intracellular signalling processes
  • alternatively , receptors may be or control ion channels, leading to altered levels of intracellular ions
  • 2nd messengers include: inositol trisphosphate (IP3), diacylglycerol (DAG), cyclic nucleotides (e.g. cAMP, cGMP), calcium ions
  • intracellular signalling proteins alter activity of target proteins resulting in changes of cell behaviour (response)
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27
Q

Describe a signalling cascade

A
  • at each step in a signalling cascade, the “product” of one step becomes the activator or substrate of the next
  • amplification may occur e.g. if a kinase phosphorylate multiple substrate molecules
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28
Q

What does the strength of a receptor signal depend on?

A
  • amplification e.g. via activities of downstream kinases or downstream enzymes
  • attenuation (limit magnitude of signal - de amplification): phosphatases, counter-movement of ions, 2nd messenger breakdown; GTPase activating proteins
  • availability of scaffolding proteins e.g. AKAPs, PDZ proteins : bring components of signalling cascades into close proximity; increased local concentrations of soluble factors; temporal focusing via negative regulators
  • tachyphylaxis/ desensitization: acute - ion channel conformation, receptor phosphorylation; substrate depletion; receptor internalization, degradation; receptor mRNA down regulation
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29
Q

What does Pleiotropy mean?

A

One signal can elicit multiple outcomes
- GPCR activation may trigger multiple signalling cascades, e.g. in transcription, ion channel activity, cell proliferation, cell survival
- receptor has choices of the pathway it triggers for example

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30
Q

What does convergence mean?

A

Multiple signals can activate a common outcome
- RTKs (receptor tyrosine kinase) activate PI3 kinase via p85 (phosphorylates)
- GPCRs activate pI3 kinase via p101
- p85 and p101 have different preferences for PI3 subtypes
- in spite of functional overlap, RTK and GPCR signals may produce different outcomes

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31
Q

What is signal integration?

A

Protein is activated only when both sites are phosphorylated. i.e. activated when signals A and B are present simultaneously - inhibiting one or the other signal may be sufficient to block biological function

32
Q

What are example of when cell communication goes wrong?

A
  1. Losing the signal (type I diabetes)
  2. When target ignores the signal (type II diabetes)
33
Q

Describe type I diabetes

A
  • Normal blood sugar regulation: food enters body, food broken down, sugar enters bloodstream, sugar stimulates cells in pancreas to release insulin, insulin travels through blood stimulates liver, muscle and fat to take up sugar for later use
  • In type I diabetes: pancreatic cells that produce insulin are lost; insulin signal is also lost: sugar accumulates to toxic levels in blood (loss of control of extracellular glucose levels)
    Without treatment, diabetes can lead to kidney failure, blindness, and heart disease later in life
    Know as juvenile diabetes- occurs in younger people typically
    Treatment involves the injection of insulin (more ligand)
  • unable to produce insulin signal (insufficient ligand)
34
Q

Describe type II diabetes

A

Do produce insulin, but target cells lose ability to respond, leading to hyperglycemia and abnormal insulin secretion
Not able to respond to insulin effectively
- end result is the same: blood sugar levels become dangerously high
- treatment for type II is glucagon-like peptide 1 receptor agonists, which lower blood glucose levels (modulates glucose release)

35
Q

Describe the receptor structure of ligand-gated ion channels

A
  • multiple subunits form a pore
  • agonists binding triggers opening
  • ionotropic receptors
36
Q

Describe the receptor structure of 7TM receptors

A
  • extracellular N-terminus, intracellular C-terminus
  • ligand binds to TM core or extracellular domain
  • intracellular domain typically couples to G proteins
    *7= can form oligomers etc, but also monomers
37
Q

Describe the receptor structure of 1TM receptors

A
  • extracellular N-terminus, intracellular C-terminus
  • ligand binds to extracellular domain
  • most have intracellular enzyme function
38
Q

Describe the receptor structure of nuclear receptors (no TM domains)

A
  • found in cytosol or nucleus
  • agonist-bound receptors function as dimers
39
Q

What is the time frame of different receptor signalling cascades?

A
  1. Ligand-gated ion channels (ionotropic receptors): milliseconds, e.g. nicotinic ACh receptor
  2. G-protein-coupled receptors (metabotropic): seconds, e.g. Muscarinic ACh receptor
  3. Kinase-linked receptors: hours (can be minutes tho), slower, e.g. cytokine receptors
  4. Nuclear receptors: hours, e.g. oestrogen receptor (dimers with a few exceptions, transcription factors)
    *All receptors have the potential to initiate slow responses, e.g. protein synthesis - all 4 can affect gene transcription as well
40
Q

Describe nuclear receptors

A
  • some signalling molecules (ligands) diffuse across membranes and bind to receptors in either cytosol or nucleus
  • ligands are hydrophobic (has to get through membrane to access intracellular receptors) lipid soluble
  • if receptor is in cytosol, receptors translocate to the nucleus after ligand binding
  • many diverse ligands, but have similar mechanism of action - regulate gene transcription
  • produce function in the nucleus
41
Q

What are the different nuclear receptor types?

A
  1. Type I - Steroid receptors
    - bind to steroid hormones
    - cytoplasmic
    - dimerize and move to nucleus upon activation (homodimeric)
    - bound to chaperones
    - chaperone dissociates upon binding/dimerization
  2. Type II - RXR heterodimers:
    - e.g. thyroid hormone R, retinoic acid R, PPARs
    - predominantly in nucleus (remain in nucleus whether in active/inactive state)
    - heterodimerization with retinoid X receptor (RXR)
  3. Type III
    - similar to type I (mechanism)
  4. Type IV
    - function as monomers or homodimers
42
Q

What is the structure of nuclear receptors?

A

3 main domains:
- AF1: transactivation domain (contain binding sites to regulate)
- DBD: DNA binding domain (recognizes particular gene sequences)
- LBD: ligand binding domain (transactivation, transrepression, dimerization - multiple actions, binds activators/repressors)

43
Q

Give an overview of nuclear receptors

A
  • ligands include steroid hormones, thyroid hormones, vitamin D and retinoic acid, as well as certain lipid-lowering and antidiabetic drugs
  • receptors are intracellular proteins, so ligands must first enter cell
  • receptors consist of a conserved DNA-binding domain attached to ligand-binding and transcriptional control domains
  • DNA-binding domain recognizes specific base sequences, thus promoting or repressing particular genes
  • pattern of gene activation depends on both cell type and nature of ligand, so effects are highly diverse
  • effects are produced as a result of altered protein synthesis and, therefore, are slow in onset
44
Q

What are enzyme-linked receptors? Groups?

A

3 main groups:
1. receptor tyrosine kinases
2. cytokine receptors lacking enzymatic activity (have to recruit kinases/enzymes)
3. natriuretic peptide receptors with guanylyl cyclase activity
- all have an extracellular N-terminal binding site and a single transmembrane domain

45
Q

Describe tyrosine kinases (RTKs)

A
  • agonist binding to extracellular domain of RTK causes two receptor molecules to associate into a dimer
  • for some RTKs, a single agonist molecule binds to both receptors
  • dimer formation brings kinase domains of each cytosolic receptor tail into contact
  • this activates kinases to phosphorylate adjacent tail on several tyrosines = trans-activation
46
Q

What is the transduction mechanisms of kinase-linked receptors: the growth factor (Ras/Raf/MAP kinase) pathway

A

Auto-trans phosphorylation of the two halves of the dimer - then recruitment of other enzymes.
Phosphorylation of Grb2 after tyrosine autophosphorylation, then activation of Ras
*heterodimerization allows functional flexibility:
- binding of epidermal growth factor and related peptides to ErbB1, ErbB3, or ErbB4 leads to homodimer or ErbB2 heterodimer formation
- ErbB2 does not bind EGF peptides and is inactive by itself
- Heterodimers have greater activity
- ErbB3 homodimers are inactive but ErbB2/ErbB3 heterodimers are the most active species

47
Q

Transduction mechanism of kinase-linked receptors: the cytokine (Jak/Stat) pathway

A

Here, the receptor lacks kinase activity and must recruit one (JAK) for downstream substrate phosphorylation to occur.

48
Q

Summary of enzyme-linked receptors

A
  • enzyme-linked receptors all share a common architecture, with a large extracellular ligand-binding domain connected via a single a-helix to the intracellular domain
  • receptors for various hormones (e.g. insulin) and growth factors are tyrosine kinases
  • cytokine receptors activate cytosolic kinases
  • signal transduction generally involves dimerization of receptors, followed by (auto) phosphorylation of tyrosine residues
  • kinase-linked receptors govern cell growth and differentiation; they also act indirectly by regulating gene transcription
  • a few hormone receptors (e.g. NPR-A) have a similar architecture and are linked to guanylate cyclase
49
Q

Describe ligand gated ion channels (ionotropic receptors)

A

Convert chemical signals into electrical ones
- rapid signalling in electrical excitable cells e.g. nerve cells, muscle
- ligand binding transiently opens or closes ion channel leading to brief changes in ion permeability across plasma membrane and excitability of target cell
- ligand-gated ion channels, like other types, are selective for which ions they allow across the plasma membrane

50
Q

Describe the ligand-gated ion channel structure

A
  • LGICs may be made up of both agonist-binding and non-binding subunits. The combination of subunits may vary, which can give rise to multiple receptor subtypes
    1. many LGICs belong to the Cys-loop superfamily of transmitter-gated ion chanels. These are pentamers or tetramers of 4 TM-spanning subunits, where both the C- and N-termini are extracellular
  • 5HT3 receptors
  • nicotinic acetylcholine receptors
  • GABAa receptors
  • glycine receptors
    2. ionotropic glutamate receptors are tetramers where each subunit has an extracellular N-terminal domain, three transmembrane, a channel lining re-entrant ‘p-loop’ and an intracellular C-terminal domain
    3. P2X purinergic receptors consist of trimers of 2 TM subunits, each with intracellular C- and N-termini and a large extracellular loop
51
Q

Describe the LGIC function and ion selectivity

A
  • GABAa and glycine receptors are anion-selective (chloride channels) and decrease excitability
  • other LGICs are cation selective (Na, K, Ca) and tend to be excitatory
  • some isoforms show selectivity among cations, e.g. AMPA and kainate receptors are relatively impermeable to Ca under normal conditions
  • channel opening in response to agonist binding can be sensitive to the number of binding site occupied e.g. multiple binding domains and all have to be occupied in order to be activated (concerted channel opening) - other cases can have partial opening depending on how many binding sites are occupied (subunit specific channel opening)
52
Q

Describe G protein-coupled receptors

A

7 transmembrane spanning proteins
- largest family of receptor proteins (encoded by more than 3% of genome)
- ~ 800 genes encode GPCRs in humans
- an estimated 30-50% of all prescribed drugs target GPCRs
- interaction between receptor and target protein mediated by G protein
- binding of ligand to receptor leads to alteration in: 1. concentration of intracellular mediators, or 2. ion permeability at plasma membrane
- indirectly regulate activity of other membrane proteins (enzymes, ion channels) or intracellular proteins

53
Q

Describe the GPCR structure

A
  • extracellular N-terminus
  • central core of 7 transmembrane helices (TM-I to -VII or H1-H7)
  • 3 extracellular loops (e1, e2, e3)
  • 3 intracellular loops (i1, i2, i3)
  • intracellular C-terminus
    *alpha helical in nature - almost v-shaped, have proline-helix breaker = kink
54
Q

G protein-mediated signalling overview

A
  • GPCRs are 7TM-spanning proteins; sometimes called metabotropic receptors
  • can function as monomers (usually, unless requires more subunits) but can also exist as homo-oligomers and heteromers
  • the G-protein is a membrane-associated protein comprising three subunits (GaBy), the Ga-subunit possessing GTPase activity
  • when the trimer binds to antagonist-occupied receptor, the Ga-subunit is thought to dissociate and the activate an effector (a membrane enzyme or ion channel) - alpha changes conformation, comes off By, allows them to have their effect
  • “activated” GBy-subunit can also contribute to signalling
  • activation of the effector is terminated when the bound GTP molecule is hydrolysed, which allows the Ga-subunit to recombine with GBy
  • there are multiple types of G-protein, which interact with different receptors and control different effectors
  • effectors include adenylyl cyclase, phospholipase C, RhoGEFs, and ion channels
  • multiple intracellular components govern GPCR-initiated signals
55
Q

Describe the diversity and classification of GPCRs

A
  • the superfamily of G-protein coupled receptors is one of the largest families of protein in human genomes
  • ~800 GPCR genes have been identified in humans, there are also many pseudogenes (encoding incomplete or nonfunctional proteins) as well as many full length “orphan” 7TM proteins whose endogenous (don’t know the activation) ligands are unknown and which are potential new drug targets (only half are targeted currently)
  • based on sequence similarities GPCRs can be divided into several classes, each with characteristic highly conserved residues distributed throughout the molecule
  • three main GPCR classes:
    1. A, rhodopsin-like (largest class)
    2. B, secretin-like (GRAs)
    3. C, glutamate-like
  • easily recognized when comparing their amino-acid sequences - structures are highly conserved within classes
  • nonwithstanding their similar topologies, receptors from different classes share little or no sequence similarity, a remarkable example of molecular convergence (convergent evolution - similar function although they don’t have common ancestor)
56
Q

Describe class A GPCRS

A
  • rhodopsin-like, all contain E/DRY motif at the cytoplasmic end of the third transmembrane domain and prolines at specific positions in helices 5, 6, and 7
  • includes most GPCRs and can be subdivided into at least 3 groups:
    1. Group 1a: GPCRs for small ligands including adrenergic, muscarinic, serotonergic, and histamine receptors. The binding site is buried in between the transmembrane domain (separate and ligand tucks into membrane in receptor)
    2. Group 1b: GPCRs for peptides. The binding sites for these include N-terminal, the extracellular loops and the transmembrane domains
    3. Group 1c: GPCRs for glycoprotein hormones (large). Characterized by large extracellular domains and mostly extracellular binding sites (longer extracellular amino domain)
57
Q

Describe the “ionic lock” in B2-adrenergic receptors

A

Activation of B2-adrenergic receptor involves disruption of an ionic lock between Asp130/Arf131 in TM3 and Glu268 in TM6.
This “ionic lock” normally prevents movement in H6 relative to H3; mutation results in spontaneous activation.

58
Q

What is the two-state model of receptor activation?

A
  • receptors isomerize between resting (R) and active (R*) conformations (increased attracted state)
  • agonist binding is not required to generate R* (R* state will signal no matter what, just signals more with agonist) - spontaneous receptor activity
  • agonists bind with higher affinity to and also promote isomerization to R* (prevents going back to R)
  • this key initial step defines the drug-receptor interaction, and it typically followed by a biochemical signalling cascade that leads to an ultimate cellular response
  • an antagonist will bind with equal affinity to both R and R*, while an inverse agonist (tends to drive more to ground state) will bind with higher affinity to the resting state. Either will competitively inhibit the effects of an endogenous agonist in vivo.
  • agonist binding promotes rearrangement of transmembrane helices - TM3 and TM6 move apart
  • this conformational change enables receptor to activate G protein
59
Q

Describe the class B GPCRs

A
  • secretin receptor-like GPCRs
  • significant homology between class B hormones (that activate receptors)
  • “hot dog in a bun” binding mechanism where the peptide agonist interacts with both the N-terminal extracellular domain and also the transmembrane domains
  • all class B GPCRs activate Gs/adenylyl cyclases, and some can couple to other pathways as well
  • not as easy to target with drugs - not as tight of binding as class A
  • e.g. secretin, calcitonin, adrenomedulin, urocortin, amylin, glucagon, etc.
60
Q

What are Receptor Activity Modulating Proteins (RAMPs)?

A
  • class B interacts with them
  • ligand binding selectivity of some class B receptors is altered by the binding of RAMPs (bind in parallel next to TM7)
  • calcitonin receptor when bound to RAMP1 or RAMP3 changes its binding preference to amylin (i.e. it becomes an “amylin receptor”)
  • depending on which RAMP it associates with, calcitonin receptor-like receptor (CRLR) can bind selectively to either calcitonin gene related peptide (CRLR + RAMP1) or adrenomedullin (CRLR + RAMP2 or RAMP3)
  • act as chaperones
61
Q

Describe adhesion GPCRs

A
  • formerly considered a subgroup of class B
  • large N-terminal domain that may bind directly to other cells or to the extracellular matrix
  • N termini of these proteins might mediate cell-to-cell adhesion (and cell migration) either by binding to components in the extracellular matric or by interacting with membrane proteins on other cells
  • cleavage of GPCR autoproteolysis-inducing (GAIN) domain causes receptor activation
  • large extracellular domain
62
Q

Describe class C GPCRs

A
  • glutamate receptor-like including mGluRs, Ca sensing receptors and GABA receptors
  • also include sweet and umami T1 taste receptors (savoury flavours)
  • “venus flytrap” binding mechanism (pakmen)
  • small activating molecule binds to N-terminus (large)
  • leads to conformational changes in the N-terminus result in activation of transmembrane domain of receptor
  • function as dimers
63
Q

Describe GABAb receptors

A
  • requisite dimers
    GABAb1: binds agonist but does not signal to G protein. cannot traffic to PM unless co-expressed with GABAb2
    GABAb2: signals but does not bind
  • all class C GPCRs thought to function as dimers
64
Q

What is the frizzled receptors?

A
  • 11 genes
  • frizzled receptors respond to WNT proteins; these are involved in cell-to-cell signalling during many developmental processes
  • the canonical WNT/frizzled-beta-catanin pathway does not appear to require G proteins
65
Q

What are taste 2 receptors?

A
  • recognize bitter chemicals - protection from poisons
  • humans have 28 T2R genes and 16 pseudogenes
  • rats have 37 T2R genes and 7 pseudogenes
  • T2R polymorphisms may be responsible for individual differences/ aversions (able to taste the bitter taste of grapefruits - hate it)
66
Q

Describe G protein subunits and subfamilies

A
  • G proteins are identified by their Ga subunits
  • four Ga subfamilies:
    1. Gas (Gasl, Gass, Gaolf; long, short, olfactory)
    2. Gai/o (largest: Gai1, Gai2, Gai3, Gao1, Gao2, Gaz, Gat(r), Gat(c), Gagust)
    3. Gaq (Gaq, Ga11, Ga14, Ga15/16)
    4. Ga12/13 (Ga12, Ga13)
  • five different GB genes: GB1, GB2, GB3 and GB4 have similar properties (interchangeable). GB5 differs structurally and does not interact well with Gy (gamma) - special functions
  • twelve different Gy genes: GB and Gy together form a stable dimer (function as a unit). some known selectivity, e.g. Gy1 in visual system
  • > 1000 different potential Ga-GB-Gy combinations (basis of signalling is poorly understood)
67
Q

Describe G protein effectors regulation

A
  • Gas subfamily activates adenylyl cyclase
  • Gai inhibits adenylyl cyclase, activates phosphodiesterase (Gat)
  • Gaq activates PLCB (phospholipase C), p63RhoGEF
  • Ga12/13 activates multiple RhoGEFs
  • GBy regulates AC, PLCB, voltage-gated Ca channels, inwardly rectifying K channels, p110y/PI3kinase, others
  • multiple G-mediated mechanisms of MAP kinase activation
  • intracellular Ga proteins are important in asymmetric cell division
    *some effectors are regulated by multiple G protein subtypes/subunits
68
Q

Describe adenylyl cyclase

A
  • consists of alternating hydrophobic and hydrophilic domains
  • hydrophobic domains each contain 6 TM spanning a-helices (2= 12 transmembrane domains)
  • hydrophilic regions work together to perform catalytic function (cyclization of ATP - cAMP + PPi): C1 and C2 are the catalytic domains (if you isolate, can actually produce function without the rest of the protein - main part)
  • multiple modes of AC and cAMP regulation imply intricate cellular control of this second messenger
  • cAMP binds to multiple intracellular targets and can produce both acute and prolonged changes in cell behaviour
69
Q

Describe the regulation of adenylyl cyclase

A
  • 9 different Gas-activated isoforms of adenylyl cyclase (AC1-AC9)
  • not all appear to be inhibited by Gai prtoeins, some are inhibited by GBy, whereas other are stimulated
  • decreased activity with PKA phosphorylation consistent with negative feedback
  • effects of PKC, calcium and calmodulin indicate possible indirect routes of regulation (i.e. no contact between AC and G protein)
70
Q

What is the structure of PKA?

A
  • PKA is a serine/threonine kinase composed of two catalytic (C) subunits that are held in an inactive state by association with a regulatory (R) subunit dimer
  • 3 different C and 4 different R isoforms
  • two major forms of the heterotetrameric PKA holoenzyme exist: type I and type II
  • type I PKA is predominantly cytoplasmic, whereas type II PKA associates with specific cellular structures and organelles (soluble)
  • discrete localization of type II PKA within the cell due to association of regulatory subunit (RRII or RIIb isoforms) with A kinase anchoring proteins (AKAPs) (associated with membrane) - direct location of type II to particular regions of the cells
71
Q

Cyclic AMP-dependent protein kinase (PKA)

A
  • inactive PKA consists of a heterotetrameric complex with 2 catalytic subunits and 2 regulatory subunits
  • increase in cAMP in response to activation of adenylyl cyclase
  • 2 cAMP molecules bind to each regulatory subunit leading to conformational rearrangement and dissociation from the complex (4 binding sites total)
  • substrate phosphorylation
  • PKA phosphorylates receptors, ion channels, enzymes, CREB, others
  • transcription factor CREB = cAMP response element binding protein
  • phosphorylated CREB binds to DNA binding elements called CRE (cAMP response element)
  • regulation of gene transcription
  • activates CREB
72
Q

Other cAMP-activated pathways

A
  • cyclic nucleotide-gated ion channels (e.g., in pacemaker cells of heart)
  • Epac (exchange protein activated by cAMP) activates Ras-like small GTP-binding proteins Rap1 and Rap2 and promotes cAMP-dependent exocytosis
73
Q

What is transducin?

A
  • (Gat) a specialized member of the Gai/o family
  • in the retina, cGMP-gated channel lets in Na and Ca
  • activation of visual GPCR rhodopsin by light triggers activation of Gat
  • Gat turns on PDE6, a cGMP-selective phosphodiesterase
  • as concentration of cGMP decreases, cation channels close, causing hyper-polarization of cell
74
Q

Describe the Gi/o effector pathway

A
  • inhibition of some adenylyl cyclase isoforms
  • most Gai/o inactivated by pertussis toxin (PTX, a useful tool to dissect signalling pathways)
  • GPCR-triggered signalling pathways lost with Gi/o pathways lost with Gi/o knockout or PTX include: suppression of N-type or L-type Ca channel opening, inwardly rectifying K channel activation, activation of PLCB, these appear to be largely or wholly mediated via GBy
75
Q

Describe activation of Rho signalling by G proteins

A
  • activation of Rho GTPases - cytoskeletal changes
  • Ga12/13 activates: p115-RhoGEF, PSD-95/Disc-large/ZO-1-homology-RhoGEF (PDZ-RhoGEF), leukemia-associated RhoGEF (LARG), lymphoid blast crisis-RhoGEF (Lbc-RhoGEF)
  • all GPCRs that activate Ga12/13 also appear to couple to at least one other protein subfamily
  • Gaq/11 activates p63RhoGEF; limited evidence for Gq interactions with others listed above
  • evidence that GBy can activate GEFs for Rho-like small G proteins
76
Q

Describe the Gq effector pathway

A

Gq: activates phospholipase CB (PLCB), which hydrolyzes the membrane phospholipid phosphatidyl inositol bisphosphate (PIP2) to produce two different second messengers
- diacylglycerol (DAG) activates most isofroms of protein kinase C (PKCa, PKCB, PKCy, PKC, etc)
- inosotol trisphosphate (IP3) promotes the release of Ca from SR/ER stores: Ca activates PKCa, PKCB and PKCy, increased cytosolic Ca can produce multiple other effects

77
Q

What are the G protein effects on intracellular calcium?

A
  • IP3 produced by phospholipase CB binds to ER/SR IP3 receptors to release calcium
  • cyclic nucleotide-gated ion channels permit Ca influx in retinal, olfactory, and cardiac pacemaker cells
  • PKA phosphorylates L-type Ca channels and increases intracellular calcium: GBy inhibits voltage-gated calcium channels

Cellular Physiology (7 decks)

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