GPCRs Flashcards
What are the 3 superfamilies of transmembrane receptors?
ligand-gated ion channels
GPCRs
Kinase-linked receptors
What are the 5 main Human GPCR families and some of their features?
Adhesion - look like adhesions but don’t act as such
Secretin - sm. amino terminus
Glutamate - extracellular binds to ligands
Frizzled/TAS2 - Wnt receptors control cell fate, proliferation
Rhodopsin - large group with alpha, beta, gamma and delta
What do the a, b and delta relate to in the rhodopsin family?
a - prostaglandins, dopamine, muscarinic receptors, opsin
b - peptide receptors
delta - olfactory receptors
How can GPCR chimeras be used to identify their functional regions? Give examples
with a FSH extracellular region and a LH intracellular region the expressed signalling pathway is LH.
the transmembrane domain is not important, it is the 3rd intracellular loop onwards which determines its interaction with the G-Protein
What are the subunits involved in a G Protein?
a, b, gamma
How is GDP exchanged for a GTP?
the active receptor acts as a guanine nucleotide exchange factor stimulating the exchange of GDP in the a-subunit and allowing a subunit dissociation from the beta-gamma subunit
How does GTP activate the G-Protein?
within the G-protein a short loop is held on the surface, which interacts with the terminal phosphate on the GTP. This causes subunit dissociation. When GDP is there, the phosphate cannot interact with the loop allowing the subunits to associate
How does GTP hydrolysis occur?
by nucleophilic attack by a water molecule
How is switch domain II involved in GTP hydrolysis?
it contains a conserved glutamine molecule which positions the attacking water molecule next to the GTP active site
How does the b-subunit help provide a stable support unit for the a-GDP subunit?
it has a WD repeat sequence that binds Ga in the GDP bound form
Why is Mg usually an essential co-factor for GTPase activity?
It helps form salt bridges as the +Mg and -PO group allowing the phosphate to be cleaved
Cholera toxin can render this useless
What are the G Protein families and their associated actions?
Gs - B adrenoceptors/olfactory - increase AC
Gi - a2 adrenoceptors/5HT/taste - decrease AC/cAMP
Gq - a1 adrenoceptors/5HT2 - increase PLC
What are the key players in the signalling cascade and their function?
AC - adenylyl cyclase - enzyme creating cAMP
cAMP - signal modulator for other molecules such as PKA
RGS - inhibits the Ga and therefore the signalling mechanism
PDE - breaks down cAMP
CREB - makes changes to gene expression from PKA signalling
What are the four components of AC?
receptors for hormones that regulate cAMP synthesis
regulatory G proteins
Adenylyl cyclises responsible for synthesis of cAMP
PDE enzymes responsible for cAMP hydrolysis
What does cAMP interact with?
PKA
Epac
cyclic gated nucleic ion channels
How is PKA regulated by cAMP?
it consists of 2 regulatory subunits and 2 catalytic subunits
cAMP interacts with the 2 regulatory allowing the catalytic subunits to phosphorylate stuff
What is the role of PDE?
to break down cAMP to 5’AMP
What may modulate AC isoforms?
Gia subunits
bg subunits
intracellular Ca or Ca-CaM
What are the regulatory features of Acs 1,3,8?
activated by Ca-CaM
inhibited by Gai and Gbg(type 1 only)
How are ACs 2 and 7 regulated/?
unaffected by Ca-CaM
stimulated by Gbg and PKC
what are the regulatroy feature of AC 5 and 6?
unaffected by Ca-CaM and Gbg
inhibited by Gai and free Ca and PKA
What is the regulatory feature of AC 9?
inhibited by Gai, Ca-CaM and PKC
unaffected by Gbg
Where does PKA phosphorylate in a cAMP dependent manner?
acts as a ser/thr kinase involving a covalent modification forming a phosphate ester between the phosphate group and the OH groups of the Ser/Thr side change
What is the consensus sequence for PKA phosphorylation?
NH2 —Arg-Arg-X-Ser/Thr-X
How is phosphatidylinositol involved in GPCR signalling?
kinases phosphorylate it to PIP2 and then it is cleaved by PLC
What is the result of cleaving PIP2 with PLC?
results in two second messengers
DAG and IP3
What is required for PLC activity?
Ca
How is PKC activated by DAG?
it works with Ca to activate PKC which phosphorylates several cellular proteins - in heart this can increase contractility
What are some features of DAG?
remains membrane associated and activates PKC
What 3 kinds of PKC exist?
conventional - Ca-dependent, DAG activated
novel - Ca-independent, DAG activated
atypical - Ca-independent, DAG non-responsive
What are some activators and inhibitors of PKC?
activators - phorbolesters
inhibitors - H1 and calphostin C
How is Ca released in the cell?
released by IP3 receptors in the ER membrane
signal turn off includes removal of Ca by Ca-ATPase and degradation of IP3
How is IP3 inactivated?
dephosphorylation by enzyme catalysed hydrolysis
