PHYSL 371: Oct 5-7

Dr. Kar

Neurotransmitters

1. are made and released from neurons

2. interacts with receptors to produce neurons

3. are terminated by an active mechanism

Neuromodulators

messengers that dampen/ amplify the ongoing synaptic responses

What’s synthesized in the cell soma?

neuromodulators

What has a faster response?

neurotransmitters

What is terminated by enzymes?

neuromodulators

What are receptors?

are protein structures that bind to ligands and produce a cellular response

What are intracellular receptors?

found within the nucleus or cytoplasm

• can cross the cell membrane

  • ie) lipid-soluble steroid hormones

• influence gene expression by binding to specific DNA sequences

What are cell-surface receptors?

are embedded in the plasma membrane

• bind to ligands that can’t cross the membrane

• triggers a signal transduction

What are GPCR receptors?

are metabotropic and triggers a cascade of intracellular signals

• via interactions with intermediate G proteins

• has a slower response

How do GPCR receptors work?

1. NT blinds to receptor

2. conformational change

3. activates G protein attached inside

4. activates another target protein

5. causes cell response

What are ligand-gated receptors?

are inotropic and open/ close in response to ligand binding → allows flow of specific ions

• are a multi-subunit complex that forms a pore

• has a faster response

What are enzyme-linked receptors?

are represented by growth factor receptors

• possess intrinsic enzymatic activity

• activation can lead to phosphorylation

• has a slower response

GPCR classification: Glutamate

include metabotropic receptors for glutamate and GABA

• perception of sweet and savoury tastes

GPCR classification: Rhodopsin

include receptors for biogenic amines/ peptides/ lipid

• involved in sensory stimuli like light and odour

GPCR classification: Adhesion

contain large extracellular amino-terminal domains (help it interact with molecules outside membrane)

• have an immunological function

• possess auto proteolysis-inducing domains (can self-cleave)

GPCR classification: Frizzled/ Taste2

important role in developmental biology

• help with cell development and signalling

• perception of bitter tastes

GPCR classification: Secretin

play a endocrine and metabolic role

• bind peptide hormones

• regulate digestion and hormone signalling

What is a GPCR structure like?

have 7 transmembrane-spanning α-helicies

• inside C-terminal (less variance) and outside N-terminal (more variance)

• has both intracellular and extracellular loops

Which loops are are classified as hydrophilic?

are intracellular

• ICL1, ICL2, ICL3

Which loops are are classified as hydrophobic?

are extracellular

• ECL1, ECL2, ECL3

How does a GPCR activate G-Proteins?

agonists binds → conformation change → 3rd intracellular loop interacts and activate G protein

How do GPCR binding sites help bind ligands?

membrane forms a pocket

• contains polar and charged amino acids

  • interact and bind the ligand

How does G-protein coupling affect GPCR affinity?

• G protein coupled = high agonist affinity

• G protein dissociates = low affinity

What are heterotrimeric G proteins?

composed of a Gα subunit and a Gβγ dimer

• the β and γ subunits are anchored to the plasma membrane by lipids

How are heterotrimeric G proteins activated?

1. inactive → Gα-GDP + Gβγ

2. agonist stimulation → change shape and causes GTP to physically displaces GDP

3. active → Gα-GTP, causes dissociation of Gβγ

4. cellular response → both Gα and Gβγ can interact with effectors (other proteins)

How are G-protein signalling terminated?

after cellular response

• Gα has its own GTPase activity

  • GTP → GDP via hydrolyzation

• becomes inactive and rejoins Gβγ

Heterotrimeric G proteins: Gαs

stimulates adenylyl cyclase activity

• increases cAMP production → speeds things up

Heterotrimeric G proteins: Gαi

blocks adenylyl cyclase activity

• decreases cAMP production → slows things down

Heterotrimeric G proteins: Gαq₁₁

activates phospholipase C

• hydrolyze PIP2 into DAG and IP3

  • DAG = activates PKC and causes vasoconstriction

  • IP3 = increases Ca²⁺ levels

Heterotrimeric G proteins: Gαq_12/13

acts as a controller for structural changes and targets regulator of G proteins

• turns on helper proteins RhoGEF

  • which activates GTPase Rho → cell movement

Heterotrimeric G proteins: Gβγ

controls ion channels

• regulates K⁺ channels and Ca²⁺ channels

  • helps PLP C, ADC, and PK3

What are full agonists?

ligands that shift the equilibrium in favours of active state and achieve maximal activity

What are inverse agonists?

ligands that shift the equilibrium in favours of inactive state and decrease basal activity

• aka antagonists

What are neutral antagonists?

ligands that do not effect the equilibrium and suppress agonists effects

How are GPCRs desensitized after activation?

After GPCR are activated → kinases phosphorylate serine/threonine sites on the GPCR’s intracellular loops and C-terminal tail

• reduces receptor signalling by lower ability to activate to G-proteins

What is homologous desensitization?

An agonist activates a GPCR, causing GPCR kinases (GRKs) (type of enzymes) to phosphorylate that receptor

• prevents receptor from activating G-protein → reduce its response

What are GPCR kinases (GRKs)?

Type of enzyme that phosphorylate activated GPCRs to reduce signalling

What are GRK1 and GRK7?

visual GRK

• restricted to retinal cells

What are GRK2 and GRK3?

associated with β-adrenergic receptors

• found throughout the body

What are GRK4?

mainly found in the testes

What are GRK5 and GRK6?

found throughout the body

What is heterologous desensitization?

Activation of one receptor triggers a signalling pathway → causes another receptor to become less responsive

• Mediated by second messenger-dependent kinases → PKA and PKC

  • These kinases phosphorylate receptors to reduce their signalling

How do β-arrestins terminate GPCR signalling?

GRKs phosphorylate activated GPCRs → cause β-arrestins to bind to the receptor

• This blocks the receptor from interacting with Gα proteins → preventing further G-protein signalling

What happens after β-arrestins bind to a GPCR?

β-arrestins promote clathrin-mediated internalization → bringing the receptor inside the cell. The receptor can then either:

• Recycled back to the plasma membrane

• Sent to lysosomes for degradation

How does β-arrestin high affinity affect GPCR recycling or degradation?

Receptors such as vasopressin V2 and angiotensin II AT1 tend to undergo lysosomal degradation

How does β-arrestin low affinity affect GPCR recycling or degradation?

Receptors such as β2-adrenergic receptors tend to be recycled back to the plasma membrane

What are Arrestin 1 and 4?

Visual arrestins

• mainly found in the visual sensory system

What are Arrestin 2 and 3?

Non-visual β-arrestins

• widely expressed in tissues and recruited by phosphorylated GPCRs

How do β-arrestins activate alternative signalling pathways?

Besides desensitizing GPCRs

• they can activate signalling pathways independently of G-proteins by activating various kinases

What signalling pathways can β-arrestins activate, and what are their functions?

can regulate over 20 kinases, including:

• MAPK: Regulates cell growth and proliferation

• PI3K and AKT: Regulate cell survival and growth

What is an orthosteric binding site?

where the receptor’s natural (endogenous) ligand binds

• typically on the extracellular side of a GPCR.

What is an allosteric modulator?

binds to a different site

• changing the receptor’s activity and downstream signalling

What are the three types of allosteric modulators?

1. Positive allosteric modulator (PAM): Enhances the receptor's response to an agonist

2. Negative allosteric modulator (NAM): Reduces the receptor's response to an agonist

3. Neutral allosteric ligand (NAL): Binds to the receptor but does not change its activity

What are GPCR-interacting proteins?

Proteins that bind to GPCRs and can directly mediate or modify receptor signalling, affecting how the receptor functions.

What are receptor activity-modifying proteins (RAMPs)?

are GPCR-interacting proteins that modify receptor activity and signalling

How do RAMP1 interact with calcitonin-like receptors (CLR)?

Forms CGRP receptor

• promotes vasodilation and is involved in migraines

How do RAMP2 interact with calcitonin-like receptors (CLR)?

Forms adrenomedullin 1 receptor

• promotes vasodilation.

How do RAMP3 interact with calcitonin-like receptors (CLR)?

Forms adrenomedullin 2 receptor

What other GPCRs can RAMPs interact with?

• VPAC1: Vasoactive intestinal peptide/PACAP receptor

• PTH1 and PTH2: Parathyroid hormone receptors

These interactions can modify GPCR function and signalling

What is GPCR dimerization and oligomerization?

GPCRs can function alone (monomers) or interact with other receptors to form complexes

• Homodimers/oligomers: Two or more identical GPCRs

• Heterodimers/oligomers: Two or more different GPCRs

How is the functional metabotropic GABA-B receptor formed?

Is a heterodimer made of two different subunits:

• GABA-BR1: Binds the GABA agonist

• GABA-BR2: Activates G-protein signalling

Both subunits are required for a functional receptor

How does dopamine D1/D2 heteromerization change signalling?

• D1 alone: Couples to Gs → increases cAMP

• D2 alone: Couples to Gi → decreases cAMP

• D1/D2 heteromer: Can couple to Gq/11 → increases intracellular Ca²⁺

  • promotes neuronal growth and increases BDNF expression

What are growth factors?

Are groups of protein that regulate survival, differentiation, and growth of various cells

What are the functions of insulin-like growth factors (IGFs)?

• Regulate cell and neuronal growth and survival.

• Primarily signal through tyrosine kinase-linked receptors (IGF-I, insulin, and hybrid receptors)

Family includes IGF-I, IGF-II, and insulin

How is IGF-II receptor signalling different from other IGF receptors?

it has a single transmembrane domain and has been reported to interact with G-proteins to activate downstream signalling

• doesn’t signal through tyrosine kinases