Lecture 10 GPCR 2 Flashcards
How are lipid derived 2nd messengers generated
Membrane lipids are targeted by R-regulated lipases to generate 2 different 2nd messengers
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
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
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
PIP2
Phosphatidylinositol 4,5-bisphosphate
What do the lipase enzymes also act on
PC (phosphatidylcholine) in same positions
What enzymes act on sphingomyelin
Ceramidase
Sphingomyelinase
What activates PLC
GaQ + Gbeta/gamma
What does PLC produce and what do these act on
DAG - activates PKC
IP3 - interacts with IP3R on ER to release Ca2+
What gives specificity in signalling
Selective expression and cellular localisation of signalling molecules
There are many isoforms of…
PKC
PLC
Which domain of PKC is conserved
XY catalytic
What type of kinase is PKC
Ser/Thr kinase
What is PKC structurally similar to
PKA
PKC interacts with DAG via what domain
C1
PKC interacts with Ca2+ via what domain
C2 - binds in the presence of a phospholipid
What drug mimics DAG
PMA - phorbol ester - used in research to activate PKC
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
- ATP-act channel - smooth muscle - EC ATP
- cAMP act channel - sperm - cytoplasmic cAMP
- L-type Ca2+ channel - skeletal, cardiac, brain, non - voltage
- N-type - neurones, endocrine - voltage
- P-type - purkinje neurons - voltage
- T-type - voltage, low threhhold
ER Calcium channel gating is regulated by 2nd messengers - name these 4 channels and where found etc.
- IP3receptors- Most cells including brain and smooth muscle - IP3, Ca2+
- Type I ryanodine receptor - skeletal - DHP-receptor, Ca2+
- Type II RyR - cardiac - Ca2+, cyclic adenosine diphosphate (cADP)-ribose
- 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