L7 Receptors As Drug Targets 4 – G Proteins PDF

L7 Receptors As Drug Targets 4 – G Proteins IOPPN/FOLSM 2023-24 Dr Jon Drug Design & Development Robbins 5BBM2016 Pharmacology Receptors as Drug Targets 4 – G-Prot...

L7 Receptors As Drug Targets 4 – G Proteins IOPPN/FOLSM 2023-24 Dr Jon Drug Design & Development Robbins 5BBM2016 Pharmacology Receptors as Drug Targets 4 – G-Proteins  Learning outcomes 1. Understand that heteromeric G-proteins are intracellular proteins associated with the GPCR family 2. Describe in detail how G-proteins are activated and deactivated in the G-protein cycle 3. Be able to describe which GPCRs activate which G- protein subtypes 4. Be able to describe which G-protein subtypes can activate a specific signalling pathway. Overview G-protein cycle G-protein subtypes G-protein mediated second messenger systems Toxins 2  G-proteins GTP binding proteins – Heterotrimeric (α and βγ subunits) – Homomonomeric (small single subunit) ALL – Replace GDP with GTP on activation – Hydrolyse GTP to GDP + PO4 on deactivation (GTPase activity) Beta and gamma never separate from each other, but they do separate from alpha 3  G-protein: a multifunctional protein Receptor binding βγ subunit C binding GDP binding α GTP binding N Effector binding GTPase activity & activating Shown – alpha subunit Receptor binding to C terminus Effector binding to N terminus 4  G-proteins are activated by receptors Rest state G-protein activation Subunit separation N N N βγ CC C C βγ GTP α C N βγ C GDP α GDP GTP GTP α N N More than one G-protein can be activated by a receptor 7 transmembrane domains. At rest, receptor and G protein separate Receptor activated when ligand binds and GPCR and G protein link at C terminus 5  G-protein cycle RGS = regulator of G-protein signalling 1) rest C 4) GTPase βγ +PO4 GDP α 2) binding C N GDP C GTP α 3) activation N GTP α Effector N βγ Effector βγ G protein cycle – nothing used up GTPase activity of alpha subunit converts GTP to GDP and alpha subunit reassociates with beta gamma subunit. 6  Heteromeric G-proteins 16 different types of a-subunits in mammals – Gs αs, αolf – Gi αi1, αi2, αi3, αoA, αoB, αz, αt – Gq αq, α11, α14, α15, α16 – G12 α12, α13 Note there are different subtypes of β & γ subunits Gq coupled to phospholipase C Different G-proteins couple to different receptors Receptor Subtype G-protein opsin rhodopsin Gt (transducin) muscarinic ACh M1, M3, M5 Gq & G11 muscarinic ACh M2, M4 Gi & Go adrenoceptors α1 Gq & G11 adrenoceptors α2 Gi & Go adrenoceptors β1, β2, β3 Gs purinergic PTY12, P2Y13 Gi & Go purinergic P2Y11 Gs & Gq & G11 purinergic P2Y1, 2, 4, 6, 14 Gq & G11 7  Same neurotransmitter can couple to different GPCRS and can therefore have different physiological effects. Different G-proteins couple to different effectors G-protein Effector Second messenger Gs adenylyl cyclase cAMP ↑ Gi adenylyl cyclase cAMP ↑ Gt phosphodiesterase cGMP ↑ Go calcium channel Ca entry ↑ Gq/G11 phospholipase C IP3 ↑ DA G ↑ PIP2 ↑ G12/13 Rho-GEF Rho Gq/G11 increases intracellular calcium G12/G13 further signalling 8  Quiz 1 Which family of G-proteins couple to GPCRs? Describe the activation and deactivation of a G-protein What are the 4 subfamilies of G-proteins? Which second messengers are altered by Gq? 1. Heterotrimeric G proteins 2. Ligand binds to GPCR, G protein binds to GPCR at C terminus, alpha subunit dissociates from beta gamma subunit, GDP converted to GTP by addition of phosphate. Alpha subunit acts on effector. GTPase activity hydrolyses GTP to GDP, inactivating the G protein. Alpha subunit and beta gamma subunit reassociate. 3. The four subfamilies of G proteins are Gs, Gi, Gq and G12. 4. The second messengers that are altered by Gq are IP3, DAG and PIP2. 9  βγ subunits also produce effects Activate kinases Inhibit calcium channels (desensitisation) (inhibit NT release) βγ Activate potassium channels Mop up other alpha subunits (regulate excitability) (inhibit signalling) Beta gamma subunits can produce effects including activation of kinases, inhibition of calcium channels to inhibit neurotransmitter release, activate potassium channels to regulate excitability and mop up alpha subunits to inhibit signalling. 10  Toxins that act on G-proteins Cholera toxin (CTX) – activates Gs & Gt From bacteria Pertussis toxin (PTX) – inhibits Gi & Go From bacteria Mastoparan – activates Gi From wasp venom Pasteurella multocida toxin (PMT) –activates Gq,G11,G12,G13 & Gi From bacteria Pertussis toxin causes whooping cough Some toxins act by ADP ribosylation G-protein C C N toxin ADP N NAD+ R ADP R 11  ADP molecule alters function of G protein This can activate or inhibit G-proteins C C βγ βγ CTX GTP α activates Gs GTP α NAD N N ADP-R ADP ribosylated at Arg 201 prevents GTPase activity ADP-R C C βγ βγ Go/Gi PTX GDP α GDP α inhibits NAD N N ADP ribosylated at Cys351 prevents receptor coupling Cholera toxin causes constant activation of G protein, causing abnormal excessive signalling Below = pertussis toxin – stops binding of G protein to receptor. 12  Other compounds that act on G-proteins GDP-β-S mimics GDP and prevents activation of G-proteins GTP-γ-S mimics GTP cannot be hydrolysed by GTPase activity. G-protein permanently activated AlF4- similar to GTP-γ-S, activates G-proteins YM-254890 from Chromobacterium Gq inhibitor YM-254890 is a drug synthesised from chromobacterium that inhibits Gq proteins. 13  Comparison of G-protein families Family Gs Gi/Go Gq/G11 G12/G13 Golf Gt, Ggust Gz members 2 5 5 2 receptors β-adrenoceptor M2 muscarinic M1 muscarinic chemokine 2nd mess Gs +AC Gi -AC + PLC Rho Go -VOCC Gt +PDE Toxins cholera pertussis PMT PMT PMT What happens when G-proteins go wrong Pseudohypoparathyroidism type 1a (PHP1a) with precocious puberty – Hypocalcemia – Hyperposphatemia – High parathyroid hormone (PTH) levels – Mental retardation – Signs of puberty at aged 4 14  Pseudohypoparathyroidism type 1a (PHP1a) with precocious puberty Broken down C most tissues Protein not stable Low affinity at 37oC C Arg366Ser αs for GDP GTP N αs C N Constitutively active In tissues lower GTP αs Not functional than 37oC N AC ATP cAMP Overactive Mutation in alpha subunit Arg366Ser G protein becomes unstable and does not function at normal body temperature 15  Quiz 2 1. What effects can the beta-gamma subunits have? 2. Which human diseases are caused by toxins acting on G-proteins? 3. What is ADP ribosylation? 4. Give an example of a therapeutic drug that acts on a G-protein 1. Beta gamma subunits can produce effects including activation of kinases, inhibition of calcium channels to inhibit neurotransmitter release, activate potassium channels to regulate excitability and mop up alpha subunits to inhibit signalling. 2. Cholera and whooping cough (pertussis toxin) are human diseases caused by toxins acting on G proteins. 3. ADP ribosylation is an effect produced by toxins against G proteins where ADP molecules are added to G proteins, altering their structure and thus their function. YM-254890 is a drug synthesised from chromobacterium that inhibits Gq proteins. 16  References G-proteins Neer (1995) Cell 80: 249-257. PHP1a Lania et al (2001) Eur. J. Endocrinol. 145: 543-559 17