L5 - G-protein coupled receptors (GPCRs) 1 - structure and function of GPCRs

what are G protein coupled receptors (GPCRs)?

• most diverse type of receptor family

• also known as metabotropic receptors

• a large family of membrane proteins

• they detect molecules outside of the cell and activate internal signal transduction pathways by interacting with G proteins (leading to a cellular response)

what are GPCRs indirectly linked to?

ion channels through signal transduction mechanisms such as G proteins

structural features of GPCRs

• 7 transmembrane domains (a-helices)

  —> these domains are connected together by:

  3 intracellular loops + 3 extracellular loops

• H8 domain acts as an anchor - it anchors the receptor to the membrane

• N-terminal - extracellular side - ligand binds

• C-terminal - intracellular side - G protein binds

• TM3 - centrally located next to binding pocket - key in transmitting signal from ligand binding site to inside of cell

what are the two states of GPCRs?

• active

• resting

resting state of GPCRs

• GPCR is inactive

• no ligand bound

• G protein is separate

active state of GPCRs

• ligand binds

• GPCR becomes active

• conformational change

• activates G protein

• triggers intracellular signalling

what determines what ligand can bind to GPCRs?

• the structure of the N terminal

• differences in binding pocket for the ligand

what are different classes of GPCRs determined by?

differences in the structure of the ligand binding site making them specific

what are protease-activated receptors (PAR)?

• a type of GPCR present in platelets

• N terminal of PAR acts as its own ligand

how does the N terminal of PAR act as its own ligand?

• the N terminal extracellular domain is cleaved by a protease (like thrombin)

• this cleavage exposes a new part of the N terminal that acts as a tethered ligand which then binds back onto the receptor itself to activate it

what are G proteins?

• guanine nucleotide binding protein

• belong to GTPase family

what is the function of G proteins?

• act as molecular switches inside the cell to transmit signals from the extracellular stimuli to other effectors within the cell

how are G proteins regulated?

• regulated by their ability to bind and hydrolyse GTP (ON) and GDP (OFF)

• G proteins bind GTP = ON / activated state

• hydrolyses GTP to GDP = OFF / inactivated state

what do G proteins exist as?

• heterotrimeric complexes made up of alpha, beta and gamma subunits

basic mechanism of GPCRs

1. resting state (three inactive components)

   —> GPCR inactive with G protein bound to GDP

2. ligand binds causing activation of GPCR and a conformation change (GPCR changes shape)

3. GPCR activates G alpha by exchanging GDP for GTP

4. G alpha bound to GTP and separates from G beta-gamma

5. both G alpha and G beta-gamma activate downstream effectors

6. G alpha hydrolyses GTP to GDP and then reassociates with G beta-gamma

how is G protein signalling controlled?

1. presence of ligand

2. rate of GTP hydrolysis by G alpha (GTP→GDP)

   this process can be sped up by RGS proteins

what are RGS proteins?

• Regulators of G protein signalling

• they stimulate GTPase activity in the alpha subunit

• which speeds up hydrolysis of GTP→GDP

what is the Golf a subunit?

• expressed in sensory neurons in the nose

• they bind to specific ligand of an odorant molecule

explain how an odorant molecule binding leads to an action potential being sent into the brain

1. an odorant molecule binds to the receptor

2. GTP makes the G protein active and allows it to move through the membrane

3. this then binds to an enzyme called adenylyl cyclase which uses ATP to increase levels of cAMP

4. cAMP then binds as a ligand to ligand gated ion channels - allowing influx of Na+ and Ca2+ through the channel

5. they act directly on the channel which then sends and action potential into the brain

what does the type of G alpha subunit determine?

• which downstream effector proteins will be activated or inhibited

what are the two types of effectors?

1. enzymes - generate 2nd messengers

2. ion channels - open/close to change ion flow and membrane potential

what are the two ways in which ion channel gating is regulated?

1. directly (beta-gamma subunits)

2. indirectly by 2nd messengers and their effectors

direct activation of ion channel

• signalling molecule binds to or interacts with ion channel itself

• causing it to open or close without 2nd messengers

indirect activation of ion channel

• activated G proteins regulate the activities of enzymes that control the levels of 2nd messengers

what are second messengers?

• small molecules that carry signals inside cells

what are examples of second messengers?

• hydrophobic lipids

• small soluble molecules - that diffuse through the cytoplasm (cAMP, cGMP)

• calcium ions

why is the 2nd messenger system important?

• 2nd messengers amplify the signal

• they allow for a small external signal to cause a big internal response

  (one ligand binding to one GPCR can trigger a cascade through 2nd messengers - leading to the phosphorylation (activation) of millions of proteins)

• activation of a single receptor = activation of many proteins

what are two bacteria infections that affect G proteins?

1. Cholera toxin - bacterium - vibrio cholera

2. Whooping cough - bacterium - bordatella pertussis

cholera toxin - bacterium - vibrio cholera

1. this bacterium binds to the Gs (stimulatory G protein) alpha subunit

2. this binding increase its activity which activates adenylyl cyclase which catalyses cAMP

3. this causes activation of phosphorylation events:

   increase of Cl- ions secreted

   increase of Na+ ions secreted + H2O

4. as a result there’s excess fluid + electrolytes in the lumen of the small intestine

5. leading to diarrhoea and extreme dehydration

whooping cough - bacterium - bordatella pertussis

1. affects the Gi alpha subunit (Inhibitory alpha subunit)

2. it inactivates the inhibitory nature of the G protein which results in an increase of cAMP

3. too much cAMP leads to:

   erosion of respiratory epithelium

   discharge of large quantities of mucus containing fluid

4. triggers coughing fits

what’s an example of an activating mutation in GPCRs?

• rhodopsin mutation causing night blindness

what’s an example of loss of function mutation of GPCRs?

• rhodopsin loss mutation causing retinis pigmentosa or retinal degeneration

uveal melanoma

• mutation in Gq alpha subunits

• leading to blocking of GTP hydrolysis

• which means subunits always active causing permanent signal transmission

• this activates growth pathways causing cancer to occur = melanoma