Section 3.3: Receptors

Modes of Communication b/w Cells: Contact Dependent

• e.g during development an in immune response

• a signaling molecule on the surface of one cell binds to a receptor on a neighboring cell

• requires physical contact between cells

Modes of Communication b/w Cells: Paracrine (autocrine)

• signals are released into the extracellular space and act locally on neighboring cells (paracrine)

• autocrine: the cell responds to its own secreted signal

• eg. cancer cells use this strategy to stimulate survival and proliferation

Modes of Communication b/w Cells: Synaptic

• neurons transmit signals electrically along their axons

• release neurotransmitters at synapses, which are often located far away from the neuronal cell body

Modes of Communication b/w Cells: Endocrine

• endocrine signaling depends on endocrine cells, which secrete hormones into the bloodstream for distribution throughout the body

• signaling over long distances makes use of endocrine cells

• the same types of signaling molecules are used in paracrine, synaptic and endocrine signaling; the differences lie in the speed and selectivity with whch the signals are delivered to their targets

Typical Signaling Cascade

• signal molecule binds to a cell-surface receptor

  • most signal molecules are hydrophilic and cannot cross membrane

• activated receptor triggers intracellular signaling pathways (conformational change)

  • involves a series of signaling proteins and second messengers (cAMP, cGMP, IP₃) that relay and amplify the signal

• these signaling proteins act on effector proteins, which change cell behavior

GPCR Family

• 7 TMS

• largest family of cell-surface receptor; ~350 GPCRS in humans

• bind a variety of ligands: peptides, hormones, growth factors, fatty acids, odorants, light

  • many GPCRs still have unknown ligands

G-Protein Coupled Receptor (GPCR)

• membrane receptor bound to a G-protein

• GPCRs activate a trimeric G protein on the inner membrane surface

  • G protein = ⍺, β, γ subunits

• Inactive state: G protein has GDP bound to the ⍺ subunit

• when ligand binds GPCR (activated), it acts as a GEF

  • ⍺ releases GDP → binds GTP → ⍺ dissociates from βγ → activate target enzymes or ion channels (e.g. in G₅, ⍺-GTP activates adenylyl cyclase

• G protein remains active until the ⍺ subunit hydrolyzes GTP → reassociates with βγ

  • G protein has intrinsic GTPase activity stimulated by RGS proteins (regulators of G-protein signaling)

  • RGS determine how quickly bound GTP is hydrolyzed to GDP and how long G protein remains active

• everything stays confined within the bilayer due to the lipid anchor

G-Protein Coupled Receptor (GPCR) FIGURE

GPCR Example: Adrenaline

• when released into blood: increases heart rate, raises blood pressure, opens airways in lungs, boosts blood sugar

  • mediates mobilization of energy (fight or flight)

• β2-adrenergic receptor (β2-AR) is a type of GPCR that responds to adrenaline

• epinephrine binds deep within the membrane; the binding site is formed by a.a’s from many TMSs

  • helices 3,5 and 6 participate in binding

  • the interaction is stereospecific; 3D orientation of epinephrine is critical for binding (not many things can bind in the pocket and stay there)

β2-AR: Active vs Inactive

• inactive state: bound to carazolol (inverse agonist or antagonist)

• active state: part of the β2AR-G’s complex

  • TM6 moves outward to allow G-protein binding

  • TM5 and TM3 also shift subtly to transmit the signal

Agonist

• binds GPCR and stabilizes active form → activates G-protein

Inverse Agonist

• stabilizes the inactive form of the receptor

Antagonist

• blocks receptor activation by preventing the conformational change that would activate the G protein

Adenylate Cyclase Pathway

Desensitization: β2 Arrestin

• after prolonged stimulation, β-arrestin binds to the receptor → prevents further G protein activation → receptor desensesitization

Desensitization to Adrenaline

• when epineprine is present continuously, β-adrenergic receptors respond less over time (desensitization, leading to a reduced cellular response)

  • eg. chronic stress

Key proteins:

• β-adrenergic receptor kinase (βARK): phosphorylates receptor on C-terminal

• β-arrestin: binds phosphorylated receptor → prevents further G-protein activation

Epinephrine and Synthetic Analogs

• epinephrine binds β-adrenergic receptors; affinity is measured as dissociation constant (Kd) of receptor-ligand complex

• synthetic analogs: chemically modified versions of epinephrine that can either mimic or block its action

• isoproterenol: synthetic agonist with higher affinity than epinephrine (strongly activates β-receptors)

• propranolol: synthetic antagonist (beta blocker), extremely high affinity → blocks receptor activation

Receptor Ligand Binding Interaction

• rate of formation of RL complex: k_on [R] [L]

• rate of dissociation of RL complex: k_off [RL]

• at equilibrium: rate of formation = rate of dissociation

• Kd = k_off / k_on = ([R] [L])/[RL]

  • Kd: when 50% of receptor is bound to ligand

  • low K_d → high affinity (less ligand needed to occupy 50% of receptors)

Receptor Ligand Interaction Experiment: Surface Plasmon Resonance (SPR)

• technique used to measure binding interactions in real time w/o labeling the ligand or receptor

  • produces a sensorgram, showing response (binding) vs time

• baseline: before ligand is introduced → no binding

• association phase: ligand binds receptor → signal increases

• equilibrium phase: rate of binding = rate of dissociation → plateau in signal

• dissociation phase: ligand removed → signal decreases

Receptor Ligand Interaction Experiment: Surface Plasmon Resonance (SPR) FIGURE

• Red: fast association, fast dissociation → transient binding.

• Purple: fast association, slow dissociation → strong/stable binding.

• Blue: slow association, slow dissociation → gradual, stable binding.

• Green: slow association, fast dissociation → weak, transient binding.

Receptor Ligand Interaction - Experimentation

• isolate cells (or membranes) containing the receptors. Place onto the filter

• prepare saturating amounts of ligand molecules (eg. radioactive or fluorescent)

• pass the mixture through the filter (pore small enough to retain cells or membranes)

• wash away unbound ligand molecules

• measure bound radioactivity (the sum of specific + non specific binding)

Binding Assay- Typical Curve

• cells w/ receptors: 1000-50000 copies per cell

• cells were incubated for 1 hour at 4ºC with radioactively labeled adrenaline

• assume no endocytosis of the cell is taking place

• curve A: adrenaline bound to receptors and non specifically bound (never reach a plateau)

• curve B: difference between A and C (ideal)

• this type of curve allows determination of receptor number (B_max) and Kd

CFTR (-/-) Mice are Resistant to Cholera Toxin

• people who are carriers of cystic mutation (CFTR +/-) may receive a survival advantage in diseases that cause massive salt and water loss (eg. cholera)

  • CFTR is the Cl- channel that cholera toxin hijacks to cause secretory diarrhea

• cholera forces CFTR to stay permanently open

Mouse Experiment:

• CFTR (-/-): cannot secrete chloride → cannot develop cholera diarrhea

• CF (+/-): reduced CFTR activity → less activity than normal mice

• WT (+/+): full CFTR function → strong diarrhea response to cholera

Regulation of CFTR (ABCC7) by PKA

• CFTR carries a regulatory domain (R-domain) that is phosphorylated and regulates transporter activity

  • phosphorylated = open; dephosphorylated = blocks channel gate (no Cl- flow)

• β-adrenergic signaling increases cAMP, PKA is activated and phosphorylates the R domain

cAMP

• Cyclic Adenosine Monophosphate

• intracellular second messenger molecule involved in many cell signaling pathways

• relaying signals from hormones like adrenaline to activate enzymes, open channels, and regulate genes

Vibrio Cholerae

• Vibrio cholerae is the bacterium that causes cholera; to cause disease i must deliver cholera toxin into intestinal epithelial cells

• the cholera bacterium has a large secretion system that spans the inner membrane, periplasm and outer membrane

  • this apparatus is ATP powered

  • function is to export cholera toxin out of the bacteria and into the environment/host

Pre-Ctx A/B

• precursor forms of cholera toxin, subunits A and B

• include a signal peptide that directs them thru the Sec secretion system

• cannot fold in cytosol (becomes stuck thru Sec pore → folding occurs after protein is in periplasm → delivered to secretion apparatus (the one than spans the multiple membranes and ATP powered)

Cell Penetration and Action of Cholera Toxin Part 1

• cholera toxin (CT) = AB₅ toxin (6 subunits)

• CT binds to GM1 glycosphingolipid on intestinal epithelial cell surface → toxin is endocytosed in retrograde direction (endosome → Golgi → ER)

  • CtxA contains a KDEL sequence (guides direction to ER instead of lysosome)

• in the ER, cholera toxin mimics a misfolded protein

• protein disulphide isomerase (PDI) breaks the disulfide bond that links CtxA and B

Cell Penetration and Action of Cholera Toxin Part 2

• once freed, CtxA1 is recognized as misfolded and transported to cytosol via Sec61 complex → most of CtxA1 is degraded by proteasome

• remaining fragment is enzymatically active → transfers ADP-ribose moiety of NAD⁺ to G-⍺ subunit, inactivates GTPase activity→ Gs⍺ is always active

• always active Gs⍺ → increased production of cAMP (activated adenyl cyclase) → activates protein kinase A (PKA) → CFTR phosphorylated and permanently open → massive Cl^- efflux → Na⁺ and water follow → diarrhea

Cell Penetration and Action of Cholera Toxin FIGURE

Another Cholera Toxin Figure

Anti-Diuretic Hormone (ADH): Background

• 9 a.a peptide

• in 24H, kidneys produce ~170L of primary urine, but extensive water reabsorption controls it to 1L being excreted

• the recycling machinery is possible b/c of aquaporins (AQPs) (millions in a single kidney)

• ADH (aka vasopressin, AVP) promotes the insertion of AQP2 channels to CM of renal tubular cells → increasing water reabsorption from urine

• ADH deficiency leads to diabetes insipidus (excessive urine production)

ADH: Vasopressin Receptor Signaling (V2R)

• the binding of ADH to its receptor V2R activates a G-protein coupled signaling cascade

• AVP binding → activation of V2R → activates adenylate cyclase → increased cAMP levels→ activates PKA → triggers exocytosis of vesicles containing AQP2

• increased AQP2 at CM = enhanced water reabsorbion

ADH: Vasopressin Receptor Signaling (V2R)

Muscarinic Receptor (GPCR) Background

• muscarine: acetylcholine analog

  • binds more strongly to muscarinic acetylcholine (mAChR) than acetylcholine

  • mAChR is a GPCR, coupled to G⍺i protein

• atropine antidote antagonist

Muscarinic Receptor (GPCR): Mechanism of Action in Heart Muscle

• muscarine binds mAChR

• G⍺i dissociates from Gβγ upon GTP binding → K⁺ channels open → K⁺ efflux → hyperpolarization (more negative membrane potential) → keeps voltage-gated Ca²⁺ channels closed → reduces frequency of heart muscle contraction

Muscarinic Receptor (GPCR): Termination of Signaling

• G⍺i hydrolyzes GTP→ GDP

• G⍺i-GDP recombines with Gβγ → channel closes → normal Vm restored

Muscarinic vs Nicotinic Receptors

Nicotinic ACh receptor: ligand-gated ion channel → fast depolarization → muscular contraction

Muscarinic ACh receptor: GPCR → slower, indirect effect thru G protein → muscular relaxation

Muscarinic vs Nicotinic Receptors FIGURE

Light Receptor - Rhodopsin: Anatomy of Retina

• Rods: responsible for high resolution and night vision

• Cones: color vision, 3 subtypes

• rods and cones form synpases with interconnecting neurons, which relay signals to ganglion cells → optic nerve → visual cortex

Light Receptor - Rhodopsin: Rod Cell Structure

• outer segment: contains ~1000 stacked discs with rhodopsin

  • discs are not connected to PM

• inner segment: cell body with nucleus and organelles

Rdodopsin: GPCR Light Receptor/Phototransduction Cycle

• rhodopsin in the disc membrane contains a chromophore (11- cis retinal)

GPCR activated by a photon

• photon → 11 cis isomerizes to all-trans retinal

• rhodopsin undergoes conformational change → meta rhodhopsin II (active opsin)

• meta-rhodopsin II activates transducin (Gt) by promoting GTP binding to G⍺t

• G⍺t-GTP then interacts with phosphodiersterase (PDE γ subunits)

Phototransduction Cycle FIGURE

Rhodopsin: GPCR Light Receptor: Chromophore Recycling

• all trans retinal dissociates from opsin

• enzymes convert it back to 11-cis retinal

• rebinds opsin → ready for next photon

Rhodopsin Figure

Rhodopsin: GPCR Light Receptor: cGMP gated Ion Channel in Rod Cells

• activation of PDE → PDE hydrolyzes cGMP → GMP → [cGMP] decreases

• Na⁺/Ca²⁺ channels in the rod outer segment require cGMP to stay open

  • low [cGMP] → channels close

  • rod cell hyperpolarizes → membrane potential becomes more negative

• hyperpolarization reduces neurotransmitter release

• light essentially inhibits the electrical signal

• ATP in the inner segment of the rod powers the Na⁺/K⁺ ATPase, creates a transmembrane electrical potential

cGMP gated Ion Channel in Rod Cells FIGURE

Adaptation/Desensitization of Phototransduction Pathway

Opsin phosphorylation: light-activated opsin can be phosphorylated by a rhodopsin kinase

• more light → more opsin in active state → more phosphorylation

Effect on G protein activation: phosphorylated opsin is less able to activate G⍺t (transducin)

• in a bright light, a larger amount of light is needed to generate the same signal (light adaptation)

Arrestin binding: at very high light levels, arrestin binds fully phosphorylated opsin

• opsin-arrestin complex cannot activate G⍺t at all → phototransduction temporarily halted

  • protects cell from overstimulation and saturation

Adaptation/Desensitization of Phototransduction Pathway

GPCR linked to the IP3 Pathway

• certain GPCRs activate phospholipase C (PLC)

• PLC cleaves PIP2 in the PM to generate inositol 1,4,5 trisphosphate (IP3) cytosolic messenger, and DAG membrane bound messenger

• at ER membrane, Ca²⁺ opens IP3 gated Ca²⁺ release channels (IP3 receptor)

  • Ca²⁺ stored in the ER quickly rises in cytosol

• DAG stays in PM; tgt w/ phosphatidylserine and Ca²⁺, helps activate protein kinase C (PKC)

  • PKC phosphorylaes target proteins

GPCR linked to the IP3 Pathway: Termination of the Signal

• IP3 dephosphorylated → inactivated (by specific lipid phosphatases)

• IP3 phosphorylated → form IP4 (by specific lipid kinases)

• Ca²⁺ that enters the cytosol is rapidly pumped out, mainly to exterior of the cell

GPCR linked to the IP3 Pathway: Figure

Sweet Receptor and IP3 Pathway

• sweet receptor is a GPCR on taste receptor cells

• activation occurs when a sweet molecule binds

• inositol 1,4,5 trisphophate diffuses thru the cytosol and releases Ca²⁺ from the ER by binding to an opening IP3-gated Ca²⁺ release channels