midterm 2

Question 1

RXR HETERODIMERS vs STEROID RECEPTORS

Answer 1

STEROID RECEPTORS
-Homodimers
Cytoplasmic → move to nucleus
-Bind hormone → activate

RXR heterodimers:
—Already in nucleus
—Bound to DNA
—-Often repressing

without ligand=corepressor dealcityahe stolen, compact from

Ligand causes corepressor → coactivator switch

Question 2

extraelil ligand _ RXR

Answer 2

extracell ligna
- thyroid horse
- vitamin D
- retinoid aid

Question 3

intacell ligand

Answer 3

Fatty acid, cholestoral derivatives ( NON steroidal) , toxic hcemyicals

Question 4

Nurrr1.

Answer 4

What about Nurr1?
Nurr1 is called an “orphan receptor” because:
We don’t know its endogenous ligand.
But it still has:
DNA binding domain
Ligand binding domain (structurally)
Can dimerize with RXR
So even though it’s orphan (no known ligand),
it still forms a heterodimer with RXR.
Orphan ≠ doesn’t function.
Orphan just means “no known natural ligand.”

Question 5

Non permissive hetodimer

Answer 5

Non-permissive heterodimers

Example: TR–RXR (thyroid receptor)
—-Ligand binding to TR activates.
—-Ligand binding to RXR alone does NOT activate.

So in these cases:
RXR ligand alone ≠ transcription.

** need partners ligand

Question 6

Permissive hetero dimes

Answer 6

Permissive heterodimers
Example: PPAR–RXR

Ligand to PPAR → activation

Ligand to RXR → activation

Both ligands → sometimes enhanced
activation

So yes — sometimes one ligand is enough.

Sometimes both amplify.
Sometimes only the partner matters.
It depends on the heterodimer type.
That distinction is testable

Question 7

Transfection Assay (Mapping Functional Domains)

Answer 7

ransfection Assay?

These constructs include:
A reporter gene (like luciferase).
A response element upstream.
Different versions of the receptor.
Then we measure reporter activity.
If transcription happens → reporter signal increases.
That’s how we map receptor function

id NOT remove the entire LBD.
They did a point mutation:
Only the amino acids that contact ligand are removed.
Corepressor binding surface is still intact.

Question 8

Disease Examples of RXR Heterodimer Receptors

Answer 8

Disease Examples of RXR Heterodimer Receptors

Thyroid Hormone Receptor (TR–RXR)
Linked to:

—Resistance to thyroid hormone
–Hyperthyroidism / hypothyroidism effects

This connects to:
Development
Metabolism
Heart rate
Brain development

🔹 Nurr1 – orphan receptor
Linked to:
Parkinson’s disease
Other neurological diseases
That’s the case study later.

Question 9

TRA and TRB

Answer 9

There are two major thyroid receptor genes:
TRα = heart
TRβ= liver, thyroid, pit , hippo

And each has isoforms (alternative splicing).

Important:
The AF-1 domain (N-terminus) differs between isoforms, but the rest of the protein is largely similar.

Question 10

Resistance to Thyroid Hormone α (RTHα)

Answer 10

Resistance to Thyroid Hormone α (RTHα)

– mutation in RTA

The Mutation
It says:
Premature stop mutation
In helix 12 of the ligand binding domain (LBD)
Very low affinity for T3 (very high Kd)
Cannot recruit coactivators
Still binds corepressors wel

** DOM NEGT

Question 11

PD iverview

Answer 11

Movement disorder

CAUSE:
-Progressive loss of dopamine-producing neurons in:
—->Substantia nigra (SN)

These neurons normally:
Refine motor output
Suppress unnecessary movement

So when they die:
Movement becomes dysregulated.

TREATMENT:
L-Dopa:
—-Dopamine precursor
—Crosses blood-brain barrier
—-Gets converted to dopamine
I
mportant detail:
Dopamine is hydrophilic.
It works via membrane receptors (GPCRs).
*only work if enough ompaimen rneurgi neuron stil laive ( WHICH CONROLLED BY NURR !)

Question 12

NUrr 1

Answer 12

Nurr1: ( OTEHR nur77, nor-1)

–is upstream of dopamine production.

–It regulates genes necessary for:
Dopamine synthesis
Dopaminergic neuron identity
Neuron survival

Question 13

NUrr 1 and its receded binding pocket

Answer 13

“Nurr1’s crowded ligand binding pocket”
Meaning:
Its LBD structure does not easily accommodate small molecule ligands.
This is why direct Nurr1 drug activation failed.
This is critical.
🧠 Now the Logic Shift
If we cannot easily activate Nurr1 directly…
But Nurr1 forms a heterodimer with RXR…
Maybe we can activate the complex by targeting RXR instead

Question 14

Bxarotone

Answer 14

Bexarotene
A high-affinity and specific RXR ligand.
So scientists thought:
“If we can’t activate Nurr1 directly,
maybe we activate the RXR side of the Nurr1–RXR heterodimer.”
That’s the workaround.

Bexarotene:
General RXR agonist
Activates many RXR complexes
Causes systemic side effects (lipids, etc.)

Question 15

BRF110

Answer 15

BRF110

-specific for RXRα
-It specifically activates ————->Nurr1–RXRα (DR%5
———>Nur77–RXRα
I
t crosses the blood–brain barrier
It is the best candidate: specific, stable, high affinity

Question 16

Experimental Proof (BRF110 Rescue Experiment)

Answer 16

They use:
Human stem cell–derived dopaminergic neurons.
Then they treat them with:
MPP+ (a neurotoxin).

Question 17

M
pP

Answer 17

What is MPP+?
MPP+ is a toxin that:
Blocks mitochondrial function
Specifically kills dopaminergic neurons
It’s commonly used to model Parkinson’s disease in the lab.
So:

What The Result Shows
With MPP+ alone:
Green signal decreases → neurons dying.
With MPP+ + BRF110:
Green signal increases → neurons rescued.

Question 18

Vasopressin Example

Answer 18

Vasopressin Example

Released when BP is low

Acts on kidney → inserts aquaporins

Acts on smooth muscle → contraction

–Same hormone.
Different cell types.
Different responses.

Why?
Because different cells express:

Different GPCR subtypes
Different G proteins
Different downstream effectors

Question 19

GPCR design principle:

Answer 19

GPCR design principle:
–Same ligand class
–Different receptor subtype
—Different G protein coupling
–Different outcome

Question 20

GLP- 1R

Answer 20

GLP-1 receptor is a GPCR.
These drugs:

—-Increase insulinsecretion
—Reduce appetite
—Slow gastric emptying

They are now among the highest revenue drugs in the world.

The chart on the slide shows explosive growth in sales over time

Question 21

Orphan GPCR

Answer 21

rphan GPCRs
It says:
There are ~100 GPCRs with no known ligands.

These are called:
Orphan GPCRs
Just like orphan nuclear receptors.
Definition reminder:
Orphan = no known endogenous ligand.

Question 22

GPCR agonist

Answer 22

NT = Actylcholien

Dopamine
norephrine

Question 23

GPCR anta gonist

Answer 23

phenoxybenzamine
propanol

Question 24

GPCR mebranetopology

Answer 24

7 Transmembrane Segments
Also called:

–Serpentine receptors
–7TMS receptors
–They snake through the membrane seven times.
–That’s universal for GPCR

orientation :

N-terminus → extracellular
C-terminus → cytosolic
Three extracellular loops (ECL1–3)
Three intracellular loops (ICL1–3)
The intracellular side is where the G protein binds.
—–>That’s critical.

Mechanistic Idea
Ligand binds outside
→ helices rearrange
→ intracellular loops change shape
→ G protein interacts
→ GDP → GTP exchange

***TM6 movement is one of the biggest conformational changes during activation.

Question 25

lign bidning difnrce gpzcr

Answer 25

Ligand Binding Differences The slide notes: Class C GPCRs → ligand binds large external N-terminus Class A GPCRs → ligand binds within transmembrane helices So binding location varies by GPCR class.

Question 26

mechanistic class a gpcr

Answer 26

Mechanistic Summary – Class A Ligand binds inside helix bundle → rearranges helices → TM6 moves outward → G protein binds → GDP → GTP exchange

Question 27

CLASS C GPCR (e.g., mGluR, CaSR)

Answer 27

🔥 Mechanistic Summary – Class C Ligand binds extracellular clamshell → clamshell closes → dimer rearranges → transmembrane helices shift → TM6 moves → G protein activated

Question 28

GEF

Answer 28

(Guanine nucleotide Exchange Factor). It does NOT hydrolyze GTP. It does NOT make GTP. **It just helps GDP fall out.

Question 29

Antagonist vs inverse agnostic

Answer 29

Specificity comes from: 1️⃣ Receptor subtype 2️⃣ Tissue expression 3️⃣ Downstream signaling context Not just the ligand. ⚪ Antagonist Blocks agonist → prevents activation, but does not change baseline activity. 🔴 Inverse agonist Reduces receptor activity below baseline, even without agonist present.

Question 30

GPCRs Grouped by Structure

Answer 30

🔵 Class A (Rhodopsin-like) Largest family (~80% of Structural features: Small extracellular N-terminus Ligand binds inside TM helix bundle Often small molecule ligands Class B Large extracellular N-terminus Peptide hormones bind partly to N-terminus Then interact with TM region Think: peptide-binding GPCRs. 🟣 Class C “clamshell” Obligatory dimers Ligand binds outside membrane Conformational change transmitted inward

Question 31

signaling gpcr to beyond

Answer 31

🧠 Step 1 – Inactive State Before ligand: GPCR is inactive. G protein = Gα–GDP + Gβγ. They are associated together at membrane. No signaling happening. Important: Gα has GDP bound. GDP = OFF state. Step 2 – Ligand Binds GPCR Ligand binds extracellularly. GPCR changes conformation. TM6 moves outward. Intracellular cavity forms. Now G protein can bind tightly. Step 3 – GPCR Acts as a GEF GPCR does NOT add GTP. It acts as a: 👉 GEF (Guanine nucleotide Exchange Factor) Meaning: It causes GDP to fall off Gα. Step 4 – GDP Leaves GDP dissociates from Gα. Now Gα has no nucleotide bound briefly. Inside the cell: [GTP] > [GDP] So GTP binds automatically. Step 5 – GTP Binding Activates Gα GTP binding causes: Conformational change in Gα Decreased affinity for Gβγ Separation of Gα–GTP from Gβγ Now BOTH can signal. This is important: Gβγ is not just passive — it can activate effectors too. Step 6 – Termination Gα has intrinsic GTPase activity. It hydrolyzes: GTP → GDP Now: Gα–GDP reassociates with Gβγ Cycle resets Signal OF

Question 32

Pathway 2: GRK + β-arrestin pathway

Answer 32

ere’s what really happens: 1. Agonist activates receptor. 2. GRK phosphorylates the receptor. 3. β-arrestin binds. Now two things happen: A) It blocks further G protein coupling (desensitization). B) It acts as a scaffold to activate its own signaling pathway (ERK cascade). So β-arrestin is NOT just “off.” It redirects signaling.

Question 33

Membrane tethering of G proteins

Answer 33

Membrane tethering of G proteins increases local concentration and accelerates receptor–G protein coupling.

Question 34

GPCR --> PKA ( stoic vs cat0

Answer 34

Summary of GPCR → PKA cascade (THIS IS HIGH YIELD) This slide literally answers “stoichiometric vs catalytic” for the pathway. (A) Signal binding GPCR = stoichiometric One ligand molecule binds one receptor and flips it to active. When ligand leaves, receptor can go back inactive. (B) Activated GPCR (as a GEF) = catalytic One activated GPCR can activate many G proteins (big amplification step) . (C) Gαs activating Adenylyl Cyclase (AC) = stoichiometric One active Gαs binds one AC to activate it. (Then AC makes lots of cAMP = catalytic, that shows up conceptually next.

Question 35

AKAP

Answer 35

AKAPAKAP = A-Kinase Anchoring Protein These proteins: * Bind the Regulatory (R) subunits of PKA * Hold PKA in specific locations inside the cel - inactiv syub unti C boun to AKPA - complex - active suvitnie releases for AJAP r subunit complex when camp binds.

Question 36

Toxins G proteins CHOLERA

Answer 36

Cholera Toxin (targets Gαs) From the slide: * Specific for Gαs * Inhibits GTPase activity * Makes GTPase permanently inactive Normally: Gαs–GTP → hydrolyzes → Gαs–GDP (turns OFF) Cholera toxin blocks the GTPase function. So: Gαs–GTP cannot hydrolyze GTP. It stays ON. That means: Adenylyl cyclase is constantly stimulated. cAMP levels become very high. This is like a stuck accelerator pedal. *** CFTR CHNAGGEL. cl enter --. water levels lead.

Question 37

pertussis toxin

Answer 37

g protine 🔴 Pertussis Toxin (targets Gαi) From the slide: * Specific for Gαi * Prevents activation by GPCR Mechanistically: Pertussis toxin prevents Gαi from interacting with GPCR. So: Gi cannot become GTP-bound. It stays inactive. That means: Gi cannot inhibit adenylyl cyclase. So AC activity increases. cAMP increases. This is like removing the brake

Question 38

Pde

Answer 38

inactivates camp

Question 39

phosphatetidylonisto ( PI)

Answer 39

- membrane lipid - minor lipid inner leaflet membrane - insotiol = head grow sugar Can be phosphorylated to generate PIP, PIP2, PIP3 - six carbon ring each carbon can be phsphryaltes - fataty acid ycajn--* inner lipid membrane

Question 40

Gas pathway

Answer 40

clear = PI ( 4,5) P2 -- DAG = activates prieitnkainase -- IP3 = release ca form ER Ligand binds GPCR → Gαq-GTP → activates PLC-β → PLC cleaves PIP2 → DAG + IP

Question 41

PKC

Answer 41

PKC has: * A catalytic domain (the part that phosphorylates) * A regulatory domain * A pseudosubstrate sequence (That pseudosubstrate is blocking the catalytic site. So PKC is OFF.) Just sitting in the cytosol. Step 1: Ca²⁺ Rises IP3 opened the ER channel. Cytosolic Ca²⁺ increases. PKC has a domain that binds Ca²⁺. When Ca²⁺ binds: PKC changes shape slightly. This allows it to move toward the membrane. But it is NOT fully active yet. Step 2: DAG Is in the Membrane Remember: PLC cleavage produced DAG. DAG is embedded in the membrane. PKC has a DAG-binding domain. When PKC reaches the membrane, it binds DAG Step 3: Membrane + DAG + Ca²⁺ Together Now three things are true: 1️⃣ Ca²⁺ is bound 2️⃣ PKC is at the membrane 3️⃣ DAG is bound This stabilizes an active conformation. The pseudosubstrate moves out of the catalytic site. Now the catalytic site is exposed. Now PKC can phosphorylate targets.

Question 42

pherbol

Answer 42

- plant derived -imilar to DAG * Bind the DAG-binding site on PKC * Mimic DAG * Strongly activate PKC * Stay bound much longer than DAG

Question 43

Combinatorial Effects OKADAIC ACID ”

Answer 43

Okadaic acid: * Inhibits serine/threonine phosphatases (especially PP1 and PP2A) So: Phosphorylated proteins cannot be dephosphorylated efficiently.

Question 44

PH domain

Answer 44

What Is a PH Domain? A PH domain is: * A protein domain * That binds specific phosphoinositides * Especially PIP2 or PIP3 It is not an enzyme. It is a targeting module.

Question 45

calmodulin

Answer 45

Calmodulin is: * A small cytosolic protein * Binds Ca²⁺ * Changes conformation when Ca²⁺ binds It is a Ca²⁺ sensor. Calmodulin has 4 Ca²⁺ binding sites. When Ca²⁺ binds: * Calmodulin changes shape * It can now bind to target proteins * It activates certain enzymes One major target: 👉 CaM Kinase II (CaMKII)

Question 46

CAMKII

Answer 46

1️⃣ Ca²⁺ rises (from IP3 pathway). 2️⃣ Ca²⁺ binds calmodulin. 3️⃣ Ca²⁺-calmodulin complex binds CaMKII. 4️⃣ CaMKII becomes active. 🔵 The Important Twist CaMKII can become autonomously active. This is the special part. When CaMKII is activated by Ca²⁺-calmodulin: It can phosphorylate itself (autophosphorylation). After autophosphorylation: It can remain active even after Ca²⁺ levels drop. * releases amoudlin \ * release CA

Question 47

PKA vs PKC vs CaMKII.

Answer 47

🔵 PKA Activated by: → cAMP Mechanism: cAMP binds regulatory subunits → catalytic subunits released. Second messenger: cAMP Does it require Ca²⁺? No. 🔴 PKC Activated by: → DAG + Ca²⁺ (for conventional PKC) Mechanism: Ca²⁺ helps membrane association. DAG binds regulatory domain. Pseudosubstrate moves out. Second messengers: DAG + Ca²⁺ 🟢 CaMKII Activated by: → Ca²⁺-calmodulin Mechanism: Ca²⁺ binds calmodulin. Calmodulin binds CaMKII. Autophosphorylation can maintain activity. Second messenger: Ca²⁺ (via calmodulin) Does not require DAG. Does not require cAMP.

Question 48

transautophsopyrlation

Answer 48

After ligand binding → receptor dimerizes. Now the two kinase domains are next to each other. They phosphorylate each other. This is called: 👉 Trans-autophosphorylation Trans = across the dimer Auto = they phosphorylate themselves (same receptor type) So: Receptor A phosphorylates receptor B Receptor B phosphorylates receptor A On tyrosine residues.

Question 49

SH2 PBD and PTYR

Answer 49

Phosphotyrosines on the tail: Are binding sites for proteins with SH2 or PTB domains. That’s how downstream signaling begins. So now the receptor becomes:

Question 50

SH2

Answer 50

bind phsophsoryttyrinse - recline AA of PTYR _ POS CAHGRE DPOCLEYS

Question 51

PTB

Answer 51

PTB domains also bind phosphotyrosine. But: They recognize sequence N-terminal to the pTyr.

Question 52

Origin of SH2 domain

Answer 52

Scientists were studying: 👉 Src (a tyrosine kinase) They noticed: Src had a region that was conserved across many signaling proteins. But they didn’t know what it did yet. That region became known as: SH2 domain

Question 53

RAS

Answer 53

What Ras Is Ras is: * A small GTP-binding protein * About 21 kDa * A monomeric G protein Monomeric means: It is just one subunit. Not αβγ like GPCR G proteins. 🔴 Why It’s Called a G Protein Because it: * Binds GDP and GTP * Switches between inactive (GDP-bound) and active (GTP-bound) Just like heterotrimeric Gα subunits. That’s the similarity.

Question 54

RAS vas G protein.

Answer 54

Similarities to Heterotrimeric Gα Ras: 1️⃣ Activated by GDP → GTP exchange (via a GEF) 2️⃣ Undergoes conformational change when GTP binds 3️⃣ Has a “switch region” that changes shape 4️⃣ Has intrinsic GTPase activity 5️⃣ Is membrane localized 6️⃣ Is activated downstream of receptors So mechanistically: It behaves like a molecular switch. 🔥 Critical Difference Heterotrimeric Gα: Has decent intrinsic GTPase activity. Ras: Has VERY weak intrinsic GTPase activity. It needs help. That help comes from: 👉 GAPs (GTPase-activating proteins). Without GAPs, Ras would stay active too long

Question 55

Why does RAS need GAPc

Answer 55

Ras Is Missing That Arginine Ras does NOT have that critical arginine. So Ras by itself: Hydrolyzes GTP very poorly. That’s why Ras needs: 👉 GAP GAP provides an “arginine finger.” That arginine inserts into Ras and helps catalyze GTP hydrolysis.

Question 56

drosophila

Answer 56

r7 mutation = phtorecption rtK huamn prolfiraito n boss--> Sev--> Drk--> Ras GEF

Question 57

Glly mutation

Answer 57

in RAS --> GTP binding pocket ( allow Gap access). Gly mutation ( to other amino acids) = CYS ---> Inhibit Gtapse active --

Question 58

SoTarasab

Answer 58

med for fly 12 mutation --> con cystein boudn ---> locks ras ---> GDP boudin ---> blocks downstream

Question 59

rout sarcoma virus

Answer 59

retrocivius ---> rea genome --> reverse trade into dNA ---> insert dna -->mutaiton

Question 60

how does the wild type SRC work

Answer 60

Src sh2 domain ---> bou by PY527 Sh3 --> stabilizes cos attach MYR ( c14) --? to membrane * inactsves until phosphorylated ----------- cancer does not have to come from viruses

Question 61

why is yh mutant SRC bad describe

Answer 61

has no tail Y527 -- cant block Sh2 -- always active

Question 62

explain effect of Y527F

Answer 62

- cant be phospholytaed - cant be unbound

Question 63

BCR abl

Answer 63

chromosome reagent --> 9-22 gene ---> encode overexerts and hyperactive --> CML

Question 64

nib

Answer 64

small drug

Question 65

mab

Answer 65

monoclonal antibodies

Question 66

Trastumab ( perception)

Answer 66

Herception breast cancer treammtnet --> HER --- monoclonal antibody --disrup recpioj dimerizaiotn -- internilciation of HER2 -- target immune cells to HEr2 * inaction, intecnilizaiton, degradation

Question 67

chronic myeloid leukemia

Answer 67

CML ----> hypoactive oncogenic kinase = BCR-abl ---> lead to cell proliferation and survival Treatment: glleevac

Question 68

Gleevac

Answer 68

kills cml cells -- inactivates BCR-abl. kinase. by binding

Question 69

Clsass A ligand

Answer 69

epinehpjroem dopamine histamine ofornaios acetylcholine Peptide * monoamine NT

Question 70

Class C ligand:

Answer 70

glutamate gaba calcium * A.A derivaties IoNs NT.