W8L3 Flashcards
Adhesion GPCRs
Is the second largest class of GPCR
Are juxtacrine receptors
▪ 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 matrix or by interacting with membrane proteins on other cells.
▪ Cleavage of GPCR autoproteolysis-inducing (GAIN) domain causes receptor activation
- N terminus will break after cleavage, but will still be attached to the receptor as a whole
Class C GPCRs
Class C - glutamate receptor-like, including mGluRs, Ca2+ sensing receptors and GABAB receptors
- mGluRs = metabotropic glutamic receptor
also include sweet and umami T1 taste receptors
“venus flytrap” binding mechanism
* Small activating molecule binds to
N-terminus
* Conformational changes in the N-
terminus result in activation of transmembrane domain of receptor
GABAB receptors are requisite dimers
GABAB1 and GABAB2 cannot function as monomers. They need to be co-expressed, which allows them to form heterodimers
Agonist binds to GABAB1, and this enables GABAB2 to activate the G protein
▪ GABAB1 binds agonist but does not signal to G protein
▪ GABAB2 signals but does not bind
▪ GABAB1 cannot traffic to PM unless co-expressed with GABAB2
▪ All Class C GPCRs thought to function as dimers too
Frizzled receptors
Frizzled receptors (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-catenin pathway does not appear to require G proteins
Taste 2 receptors
▪ 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
Heterotrimeric G protein function
Accelerated activation: GTP to GDP
Accelerated deactivation: GAP results in phosphate to be removed
Alpha subunit of G protein is bound to GDP
- when GTP dissociates (GPCR can help get GDP off of the G protein), the binding site is then open
- btw, there is more GTP than GDP
- when binding site is open, it is more likely that GTP will enter due to there being more GTP
- when GTP is bound, it enables protein signalling and effector interaction
- then, deactivation occurs due to the intrinsic ability of G protein to hydrolyze the gamma phosphate off of GTP, resulting in the GDP bound form, which is inactive state
- the deactivation step can be accelerated. There are RGF proteins that increase the intrinsic ability of the G protein to hydrolyze GTP back into GDP. This determines the duration and strength of the signalling. These are GTPase accelerating proteins (GAPs)
G protein subunits and subfamilies
G proteins are identified by their Ga subunits (G alpha)
Four Ga subfamilies:
- Gas(GasL,GasS,Gaolf)
- GasL = alpha s long
- GasS = alpha s short
- Gaolf = alpha olfactory - Gai/o (Gai1, Gai2, Gai3, Gao1, Gao2, Gaz, Gat(r), Gat(c), Gagust)
- is the largest family
- Gat(r) = rho transducing (for visual signalling)
- Gat(c) = co transducing (for visual signalling)
- Gagust = for taste - Gaq(Gaq,Ga11,Ga14,Ga15/16)
- Ga12/13 (Ga12, Ga13)
Five different Gbeta genes:
– Gb1, Gb2, Gb3 and Gb4 have similar properties
– Gb5 differs structurally and does not interact well with Ggamma
Twelve different Ggamma genes:
– Gb and Gg together form a stable dimer
– some known selectivity, e.g., Gg1 in visual system
> 1000 different potential Ga-Gb-Gg combinations
– basis of signalling specificity is poorly understood
G protein effectors
Ga = G alpha
* Gas subfamily activates adenylyl cyclase
* Gai inhibits adenylyl cyclase, activates
phosphodiesterase (Gat),
* Gaq activates PLCb (phospholipase C beta), p63RhoGEF
* Ga12/13 activates multiple RhoGEFs
Gbg = G beta gamma
- Gbg regulates AC, PLCb, voltage-gated Ca2+ channels, inwardly rectifying K+ channels, p110γ/PI3kinase, others
- Gbg signaling only via some Ga partners – why??
Multiple G-mediated mechanisms of MAP kinase
activation
Intracellular Ga proteins are important in asymmetric
cell division
Note that some effectors are regulated by multiple G protein subtypes/subunits, such as:
- adenylyl cyclase
- PLCb
- RhoGEFs
Coupling to multiple G proteins
- One type of receptor may activate more than a single type of G protein.
e.g. LPA1 receptor
- LPA binds to LPA1, which activates Gi, inhibiting AC, decreasing cAMP
- LPA binds to LPA1, which activates G12/13, activating Rho, increasing cytoskeleton rearrangement
- LPA binds to LPA1, which activates Gq, activating PLC. PLC activation causes 2 paths: IP3 activation leading to Ca2+ AND DAG activation leading to PKC
- Highly homologous receptor subtypes can link with different G-proteins which regulate separate effector species
e.g., D1 and D2 dopamine receptors
- Dopamine binds to D1, which activates Gs, activating AC, increasing cAMP
- Dopamine binds to D2, which activates Gi, inhibiting AC, decreasing cAMP
Adenylyl cyclase (AC)
- consists of alternating hydrophobic and hydrophilic domains
- has 12 TM domains
- has catalytic domains C1 and C2
- hydrophobic domains each contain 6 TM spanning a-helices
- hydrophilic regions work together to perform catalytic function
– cyclization of ATP –> cAMP + PPi - multiple modes of AC and cAMP regulation imply
intricate cellular control of this 2nd messenger - cAMP binds to multiple intracellular targets and can produce both acute and prolonged changes in cell behaviour
Regulation of adenylyl cyclase activity
Don’t need to memorize everything
- 9 different Gas-activated isoforms of adenylyl cyclase (AC1 to AC9). All are activated by Gas
- Not all appear to be inhibited by Gai proteins. Some are inhibited by Gbg, whereas others are stimulated
- AC1-3, AC 5-6 are inhibited by Gai
- AC1,3,8 are inhibited by Gbg
- AC2, 4-7 are stimulated by Gbg
- no effect on AC9 by Gbg
Calcium inhibits AC5,6,9
Calmodulin stimulates AC1,3,8
- calmodulin is influenced by calcium
- Decreased activity with PKA phosphorylation consistent with negative feedback
- PKA inhibits AC5,6,8
- PKA is activated by cyclic AMP
PKC activates AC1-3, 5, 7
PKC inhibits AC4,6
- Effects of PKC, calcium and calmodulin indicate possible indirect routes of regulation (i.e., no contact between AC and G protein)
Structure of PKA
PKA is activated by cyclic AMP
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
- when cAMP binds, it enables the activation of the catalytic subunits and they can then phosphorylate their target proteins
- there are 2 cAMP binding sites per regulatory subunit. So 4 cAMP in total on the tetramer
It is a heterotetramer: it has 2 catalytic subunits and 2 regulatory subunits
3 different C subunit and 4 different R subunit isoforms
Two major forms of the heterotetrameric PKA
holoenzyme exist: type I and type II, and they differ in cellular location
* Type I PKA is soluble and predominantly cytoplasmic, whereas type II PKA associates with specific cellular structures and organelles, and membranes
- type II regulatory subunits RII and RIIb bind to AKAPs. The AKAPs will direct the location of the type II PKAs to particular locations within the cell
* Discrete localization of type II PKA within the cell due to association of regulatory subunit (RII or RIIb isoforms) with A kinase anchoring proteins (AKAPs)
▪ inactive PK-A 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 —> substrate phosphorylation
Cyclic AMP-dependent protein kinase (PKA)
cAMP –> PKA
– PKA –> phosphorylates receptors,
ion channels, enzymes, CREB, others
– transcription factor CREB = cAMP
response element binding protein
– Phosphorylated CREB enters nucleus and binds to DNA binding elements called CRE (= cAMP response element) to act as transcription factor
– –> regulation of gene transcription
other cAMP-activated pathways
- PKA does not account for all effects of cAMP
- Cyclic nucleotide-gated ion channels (e.g., in pacemaker cells of heart)
- have intracellular binding site for cyclic nucleotides
- in pacemaker cells of heart, there is cyclic AMP gated channels
- the activation of these channels increases the rate of AP generation and speeds up the heart rate
- Epac (exchange protein activated by cAMP) activates Ras-like small GTP-binding proteins Rap1 and Rap2 and promotes cAMP-dependent exocytosis
Cyclic nucleotide phosphodiesterases
Method of controlling cAMP by turning it into non cAMP, to regulate cAMP levels
Most are selective
– cAMPselective: PDE2a, PDE3, PDE4, PDE7, PDE8,
PDE10a
– cGMP selective: PDE1a, PDE1b, PDE5a, PDE6, PDE9a
– non-selective: PDE1c, PDE11a
Various regulatory mechanisms
– calmodulin activates PDE1
– binding of cGMP to allosteric site on PDE2
increases catalytic activity
Kinase effects
– PKA increases PDE4d activity
– PKA and PKG increase PDE5 activity
– ERK decreases activity of long PDE4 variants but increases PDE4b2 activity
Interactions with scaffolding proteins: AKAPs and b-arrestins
Potentially useful therapeutic targets, but many anti-PDE drugs produce unacceptable side effects (or “surprising” ones, e.g., PDE5 inhibitor sildenafil)
