L7, Ca-signalling toolkit II

Question 1

Phospholipase C family:

Answer 1

• There are 16 members including beta, gamma, delta, eta, zeta etc
• All have a similar function -> hydrolyse PIP2 -> lipid moiety (DAG) and IP3

Question 2

Overview IP3 activation pathway

Answer 2

• Ligand activates GPCR
• G protein switches GDP for GTP, dissociates from beta gamma subunit -> activates PLC-beta
• IP3 released from DAG by hydrolysis of PIP2
• IP3 binds IP3R in ER -> opens channel -> Calcium influx from ER lumen into cytosol
• Initial release triggers CICR -> accelerates influx
• Calcium binds PKC -> migrates to membrane and binds to DAG in membrane -> activation of PKC -> phosphorylation of substrates

Question 3

Function of cADPR:

Answer 3

• Cyclic ADP ribose
• Calcium mobilising second messenger (I-C)
• Leads to release of calcium from ER through RyR (via indirect effect on SERCA pump; overloading ER with Ca2+ to trigger opening of RyR)
• Produced from NAD+ by ADP ribosyl cyclase enzyme

Question 4

Function of NAADP:

Answer 4

• Calcium mobilising second messenger
• Acts on lysosome-related organelle; activates TPC1/2 (two pore channel), triggering calcium release
• Produced from NADP by ADP ribosyl cyclase enzyme (Same enzyme as involved in cADPR production)

Question 5

Function of S1P:

Answer 5

• Sphingosine-1-phosphate
• Produced in cytosol by activity of sphingosine kinase 1 -> transported across membrane into E-C space
• -> perceived by GPCR (S1PR) -> typical IP3 pathway… -> Calcium release from ER
• Additionally, has been shown to stimulate Calcium increase independently of GPCR (pertussin toxin treated)

Question 6

How are intracellular stores of Calcium replenished:

Answer 6

• Store operated channels respond to Calcium levels in ER lumen; STIM proteins used to monitor levels

Question 7

Structure and function of STIM proteins:

Answer 7

• Found in ER membrane
• Calcium sensing domain in lumen (EF hand) -> calcium binding triggers
• CAD domains in cytosolic domain -> interact with/activate SOCs in membrane
• Upon Ca2+ binding, cytosolic domains interact with EBI (attached to microtubules) -> keeps proteins away from SOCs
• Low calcium in lumen -> STIM proteins oligomerise, can no longer interact with EBI, STIM proteins able to move into proximity with SOCs -> interaction and opening of channels -> influx of Calcium into cytosol from exterior -> pumped into ER to refill stores

Question 8

Typical sequence of OFF mechanisms during a calcium transient:

Answer 8

• Calcium buffers (highest conc., capping level, preventing global spread)
• Mitochondria
• Na+/Ca2+ exchanger
• Calcium pumps

Question 9

Key calcium efflux pathways:

Plasma membrane vs organelles

Answer 9

• In PM: PMCA (ATPase) and NCX (Na+/Ca2+ exchanger)
• In organelles: SERCA (Ca2+-ATPase), mitochondrial uniporter

Question 10

Calcium sensors:

Answer 10

• Different proteins for sensing calcium changes, including…
• Calmodulin
• Troponin C
• NCS proteins
• CaBPs
• All have EF hand domains

Question 11

Calmodulin (CaM) structure and function:

Answer 11

• 4 EF and domains
• Unbound: Open conformation
• Calcium binding -> constricts around target peptide -> downstream signalling effect (CaMKs etc.)
• Acting as molecular switches

Question 12

CaMK family:

Answer 12

• Big family with at least 4 different types in animals
• Calmodulin-dependent protein kinases
• Form vast signalling cascades
• Conserved regions: ATP binding, catalytic domain. autoinhibitory domain, Ca2+/CaM-binding domain

Question 13

CAMKII activation:

Answer 13

• Simplest version
• Inactive: constricted with A-I domain blocking catalytic domain
• Ca2+-CaM binds -> removes autoinhibitory domain from catalytic domain -> phosphorylation -> activation

Question 14

CaMKI, IV activation:

Answer 14

• More complex than CaMKII
• Ca2+/CaM partially activates; requires phosphorylation by CaMKK for full activation

Question 15

+ Annexins: Overview structure of family, comment on role

Answer 15

• Calcium dependent phospholipid-binding proteins
• All family members have a conserved repeated domain (‘annexin fold’)
• 4 domain core in all members: Includes calcium binding site, phospholipid binding site
• Binding affinity for calcium greatly increased by presence of negatively charged phospholipids; varies widely between members
• Varied effects; both up and downregulation can have pathological effects