Lecture 10 GPCR 2 Flashcards by Rosie Rudin

How are lipid derived 2nd messengers generated

Membrane lipids are targeted by R-regulated lipases to generate 2 different 2nd messengers

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What are the 2 most important 2nd messengers generated

(i) will be water soluble & diffuse through the cytoplasm eg. IP3
(ii) hydrophobic molecules, which remains in the membrane eg. DAG

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How do lipid kinases work

add phosphate groups to lipids eg. To DAG to make phosphatidic acid , or to PI to generate PIP, PIP2, PIP3

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What 3 enzymes work on PIP2 and what do they form

PLC –> breaks phosphodiester bond –> IP3 and DAG
PLD –> di-phosphate inositol and phosphatidic acid
PLA2 –> arachidonic acid

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PIP2

Phosphatidylinositol 4,5-bisphosphate

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What do the lipase enzymes also act on

PC (phosphatidylcholine) in same positions

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What enzymes act on sphingomyelin

Ceramidase

Sphingomyelinase

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What activates PLC

GaQ + Gbeta/gamma

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What does PLC produce and what do these act on

DAG - activates PKC

IP3 - interacts with IP3R on ER to release Ca2+

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What gives specificity in signalling

Selective expression and cellular localisation of signalling molecules

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There are many isoforms of…

PKC

PLC

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Which domain of PKC is conserved

XY catalytic

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What type of kinase is PKC

Ser/Thr kinase

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What is PKC structurally similar to

PKA

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PKC interacts with DAG via what domain

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PKC interacts with Ca2+ via what domain

C2 - binds in the presence of a phospholipid

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What drug mimics DAG

PMA - phorbol ester - used in research to activate PKC

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What does GFP tagging of PKC show

PKC can be used to monitor the localized activation of the enzyme in real time.

Shows agonist (for Gq) -dependent translocation to the plasma membrane

Receptors may activate multiple isoforms of PKC to bring about a coordinated change in cell function

This is transient due to DAG breakdown by lipases

How does DAG binding activate PKC

DAG binding causes dissociation of intramolecular pseudosubstrate domain from active site,

Once activated what signalling does PKC provide

Once activated PKCs can provide either Positive or Negative feedback in signaling pathway

What provides - signalling of PKC + what can this contribute to

Phosphorylation of PLCb provides negative feedback for GPCR signaling, makes signaling ‘transient’
Phosphorylation of receptors can contribute to desensitization

Imaging of DAG regulated effector(s) revealed what

Activation of GqPCRs and synthesis of DAG in the PM, recruits Munc13 (a C1 domain containing protein) to the membrane

Role of Munc 13

stimulates secretory vesicle docking to the PM, preparing it for fusion

What doesn’t involve Munc13

PKC as other fucnitons e.g. ion channel reg

PI synthesis is regulated by

many enzymes

Mutation in what gene causes Lowe syndrome

OCRL - codes for an inositol polyphosphate 5 phosphatase alter PIP2 levels

Lowe syndrome symptoms

glaucoma kidney defect mental retardation

Name 5 processes regulated by calcium signaling

``` Synaptic transmission Hormone secretion & synthesis in some cases Fertilization Muscle contraction Cytokenesis ```

Concentration in IC calcium - how

In resting cells, cytosolic [Ca2+] is kept low (~100 nM) by ATP-driven Ca2+ pumps

Receptors regulate the activity of Ca2+ channels to produce

transient rises in Ca2+

Name proteins in the lumen of the ER bind up Ca2+ to allow for high concentrations to be stored, creating a large chemical gradient

Calsequestrin and calreticulin

Striated muscle calcium pump, sequestering protein and release channel

SR Ca-ATPase Calsequestrin RyR

Smooth muscle calcium pump, sequestering protein and release channel

Ca-ATPase Calreticulin > Calsequestrin IP3/RyR

Non-muscle calcium pump, sequestering protein and release channel

One of 5 Ca-ATPase Calreticulin > Calsequestrin IP3/RyR

What regulates calcium influx at PM into cytosol

voltage gated and ligand gated channels

What regulates calcium influx at ER into cytosol

ligand gated channels

Name 6 types of PM Ca2+ channels, where found, and what controlled by

1. ATP-act channel - smooth muscle - EC ATP 2. cAMP act channel - sperm - cytoplasmic cAMP 3. L-type Ca2+ channel - skeletal, cardiac, brain, non - voltage 4. N-type - neurones, endocrine - voltage 5. P-type - purkinje neurons - voltage 6. T-type - voltage, low threhhold

ER Calcium channel gating is regulated by 2nd messengers - name these 4 channels and where found etc.

1. IP3 receptors- Most cells including brain and smooth muscle - IP3, Ca2+ 2. Type I ryanodine receptor - skeletal - DHP-receptor, Ca2+ 3. Type II RyR - cardiac - Ca2+, cyclic adenosine diphosphate (cADP)-ribose 4. Type III - smooth muscle - Ca2+, cyclic adenosine diphosphate (cADP)-ribose

Features of all RyR

Ca2+ release stimulates contraction

Information transfer is encoded

amplitude, duration and frequency of store-released calcium signaling

Information transfer is translated by

calcium-binding proteins to bring about changes in cell functions

Fast +ve feedback is

Fast positive feedback by low [Ca2+]

Fast feedback is followed by

followed by slow negative feedback elicits transient signals by Type 1 and type 2 IP3 receptors- type III receptors do not show this regulation and so produce prolonged responses that drain stores