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G Proteins - Complete USMLE Study Guide

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1. The Four Transmembrane Signaling Mechanisms (Overview)

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2. GPCR Overview - Structure and Breadth

• GPCRs represent the most numerous class of receptors
• All have a characteristic 7-TMD (heptahelical) structure
• They are the target of approximately 30-40% of all marketed drugs - extremely high clinical relevance
• They couple to the activation of one or more G proteins after ligand binding

1. Ion channel regulation (independently of second messengers)
   • GABA-B, alpha-2 adrenergic, D2 dopamine, M2 muscarinic
   • Regulate K+ conductance (typically opening GIRK = G-protein-coupled inward rectifying K+ channel)
2. Adenylyl cyclase modulation (cAMP pathway)
   • Positive: beta-2 adrenergic -> Gs -> cAMP up
   • Negative: alpha-2 adrenergic -> Gi -> cAMP down
3. PLC activation (IP3/DAG pathway)
   • Gq-coupled receptors -> PLC-beta -> PIP2 -> IP3 + DAG
4. Rhodopsin / light transduction (unique GPCR mechanism)
   • Light activates rhodopsin -> transducin (Gt) -> cGMP phosphodiesterase -> cGMP hydrolysis -> channel closure in rods

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3. G Proteins - The Basics

• They bind guanine nucleotides (GTP and GDP)
• They possess intrinsic GTPase activity (they self-terminate their own signal by hydrolyzing GTP -> GDP)

1. Heterotrimeric G proteins - the ones involved in GPCR signaling
2. Small G proteins (e.g., Ras, Rab) - monomeric, involved in growth and trafficking

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4. Heterotrimeric G Proteins

Structure

• Three subunits: alpha (α), beta (β), gamma (γ)
• The alpha subunit binds GDP/GTP and has intrinsic GTPase
• The beta-gamma complex acts as a dimer (βγ always stays together as a functional unit)

The Four Families - USMLE MUST KNOW TABLE

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5. Beta and Gamma Subunits - More Than Just Bystanders

• 5 forms (Gβ1-Gβ4 form one family; Gβ5 is separate)
• All approximately 35,000-36,000 Mr

• 11 forms, more structurally diverse
• Gγ7 is highly enriched in the striatum (USMLE has tied striatum to D2 signaling)
• Modified by isoprenylation at C-terminal cysteine residues (anchors the βγ dimer to the membrane)

• Modified by palmitoylation or myristoylation at the N-terminal domain
• These modifications determine: (a) affinity of α for βγ, and (b) whether α stays at the membrane or diffuses into cytoplasm

1. Activates GIRK channels (inward-rectifying K+ channels) - this is the PRIMARY mechanism, not free alpha
2. Inhibits voltage-gated Ca2+ channels
3. Recruits and activates GRKs (G-protein receptor kinases) to phosphorylate ligand-occupied GPCRs -> receptor desensitization
4. Activates certain subtypes of adenylyl cyclase
5. Activates the MAP-kinase / ERK pathway (via Gi-coupled receptors)
6. Interacts with phosducin, Ras-GEFs

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6. The G Protein Activation Cycle - Step by Step

• G protein heterotrimer (αβγ) is bound to the inner face of the receptor
• GDP is bound to the alpha subunit - this is the inactive state
• The receptor is loosely associated with the G protein

1. Ligand binds to the extracellular domain of the GPCR
2. Receptor undergoes a conformational change
3. This conformational change is transmitted to the alpha subunit of the G protein
4. The affinity of alpha for GDP decreases -> GDP dissociates from alpha
5. Because [GTP] >> [GDP] in the cytoplasm, GTP rapidly binds to alpha
6. GTP binding induces a second conformational change in alpha

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7. Toxins that Exploit G Protein Signaling - CRITICAL USMLE

Cholera Toxin

• Mechanism: ADP-ribosylates Gsα (adds ADP-ribose to an arginine residue)
• Effect: Permanently inhibits the intrinsic GTPase activity of Gsα
• Result: Gsα is permanently active -> constant stimulation of adenylyl cyclase -> massive ↑cAMP
• Downstream: PKA activates CFTR chloride channels in intestinal epithelium -> massive Cl- and water efflux
• Clinical: Profuse watery ("rice-water") diarrhea, severe dehydration

Pertussis Toxin (Bordetella pertussis - whooping cough)

• Mechanism: ADP-ribosylates Giα (at a cysteine residue)
• Effect: Prevents Giα from interacting with receptors -> Gi cannot be activated
• Result: No Gi inhibition of adenylyl cyclase -> net ↑cAMP
• Note: Also prevents Gi from activating GIRK channels and inhibiting Ca2+ channels
• Clinical: Pertussis (whooping cough); the toxin helps explain why airways become hyperreactive

RAS mutations (not a toxin, but same principle)

• Point mutations in Ras that impair GTPase activity (or make it constitutively GTP-bound) = locked ON
• Found in ~30% of all human cancers (K-Ras most common, especially in pancreatic, lung, colorectal cancer)

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8. Second Messenger Pathways in Detail

8a. cAMP Pathway (Gs and Gi)

1. Gs-coupled receptor activated -> free Gαs-GTP
2. Gαs directly binds and activates adenylyl cyclase
3. Adenylyl cyclase converts ATP -> cAMP
4. cAMP activates Protein Kinase A (PKA)
5. PKA phosphorylates serine/threonine residues on target proteins

• Heart: phosphorylates L-type Ca2+ channels and troponin I -> increased heart rate and contractility
• Liver: phosphorylates and activates glycogen phosphorylase (via kinase cascade) -> glycogenolysis
• Adipose: activates hormone-sensitive lipase -> lipolysis

1. Gi-coupled receptor activated -> free Gαi-GTP
2. Gαi inhibits adenylyl cyclase -> ↓cAMP -> ↓PKA activity
3. Also: free βγ from Gi opens GIRK channels (hyperpolarization) and inhibits Ca2+ channels

8b. IP3/DAG Pathway (Gq)

1. Gq-coupled receptor activated -> free Gαq-GTP
2. Gαq binds and activates PLC-beta (phospholipase C-beta)
3. PLC-beta cleaves PIP2 (phosphatidylinositol 4,5-bisphosphate) into:
   • IP3 (inositol triphosphate) - water soluble, goes to ER
   • DAG (diacylglycerol) - stays in membrane

• Binds to IP3 receptors on smooth endoplasmic reticulum (SER)
• Causes release of Ca2+ from intracellular stores
• Ca2+ then activates calmodulin and calmodulin-dependent kinases (CaMKs)

• Stays in the plasma membrane
• Together with Ca2+, activates Protein Kinase C (PKC)
• PKC phosphorylates multiple target proteins

8c. Visual Transduction (Rhodopsin/Transducin)

1. Light (not a chemical) is the stimulus
2. 11-cis-retinal (bound to opsin protein) absorbs a photon
3. Isomerization: 11-cis-retinal -> all-trans-retinal (conformational change)
4. This activates rhodopsin (the GPCR)
5. Activated rhodopsin activates transducin (Gt) - a Gi-family G protein
6. Free Gαt directly binds and activates cGMP phosphodiesterase (PDE)
7. PDE hydrolyzes cGMP -> GMP (decreasing cGMP levels)
8. Loss of cGMP causes closure of cGMP-gated cation channels in rod outer segments
9. Na+ and Ca2+ cannot enter -> rod cell hyperpolarizes
10. This decreased glutamate release signals to bipolar cells -> visual signal propagated

• Close structural homolog of transducin
• Located in taste epithelium
• Mediates signal transduction for taste
• Works via activation of a distinct form of phosphodiesterase
• This explains why retinal diseases and taste disorders can share some molecular overlap

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9. G Protein Regulation - RGS Proteins and Beyond

RGS Proteins (Regulators of G Protein Signaling)

• Analogous to GAPs (GTPase-Activating Proteins) for small G proteins
• Mechanism: Bind to Gα subunits and stimulate their intrinsic GTPase activity
• Effect: Hastens GTP hydrolysis -> faster return to inactive GDP-bound state
• Net result: RGS proteins INHIBIT G protein signaling (by speeding termination)
• All RGS proteins share a conserved RGS domain that interfaces with Gα

• Altered RGS expression implicated in hypertension, drug addiction, schizophrenia, Parkinson's disease
• This connects G protein signaling to psychiatric and neurodegenerative disease

GoLoco / Go-Loco Motif

• Found in R12 subfamily of RGS proteins (e.g., RGS12)
• Binds Gi and stabilizes the GDP-bound form of Giα
• Simultaneously causes dissociation of βγ from the G protein complex
• This releases active βγ WITHOUT receptor activation - receptor-independent signaling!
• Named because the Drosophila homolog of RGS12 is called "Loco"

AGS Proteins (Activators of G Protein Signaling)

• Structurally diverse proteins
• Act as binding partners of G protein components
• Can activate G protein signaling independent of receptor activation
• Another non-classical route for G protein activation

Phosducin

• A cytosolic phosphoprotein enriched in retina and pineal gland (also expressed in brain)
• Binds βγ subunits with high affinity
• Effect 1 (short-term): Sequesters βγ -> prevents βγ from reassociating with α -> prolongs α subunit activity
• Effect 2 (long-term): Eventually inhibits G protein signaling by preventing βγ from exerting its own direct effects AND by preventing heterotrimer regeneration
• Its ability to bind βγ is regulated by phosphorylation by:
  • cAMP-dependent PKA
  • Ca2+-dependent protein kinases

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10. GRKs - G Protein Receptor Kinases and Receptor Desensitization

1. When a GPCR is ligand-occupied and activates a G protein, free βγ is released
2. Free βγ recruits GRK from the cytoplasm to the membrane
3. GRK phosphorylates the ligand-occupied receptor (serine/threonine residues on the cytoplasmic tail)
4. Phosphorylated receptor recruits beta-arrestin
5. Beta-arrestin:
   • Uncouples the receptor from the G protein (desensitization)
   • Targets the receptor for internalization (endocytosis)
6. Internalized receptor can be recycled back to the membrane (resensitization) or degraded

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11. MAP Kinase Pathway via GPCRs

• GPCRs coupled to Gi release free βγ subunits
• These βγ subunits can activate the MAP-kinase / ERK pathway
• Mechanism: βγ can act on Ras directly or on linker proteins upstream of Ras
• Ras activates Raf (the first kinase) -> MEK (MAP kinase kinase) -> ERK (extracellular-regulated kinase)

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12. Small G Proteins

Ras Family (21 kDa)

• Originally identified as oncogene products of Harvey and Kirsten rat sarcoma viruses
• Normal cellular counterparts are proto-oncogenes
• Three mammalian Ras genes: H-ras, K-ras, N-ras (all found in brain and diverse tissues)
• All are membrane-associated (anchored by isoprenylation)

1. Growth factor receptor (RTK) is activated by its ligand
2. RTK is phosphorylated -> recruits adaptor protein Grb2 -> recruits Sos (a GEF)
3. Sos activates Ras (GDP -> GTP)
4. Active Ras binds Raf (MAP kinase kinase kinase) at its N-terminal domain
5. Raf -> MEK -> ERK -> transcription factor activation -> cell growth/proliferation

• K-Ras mutations (most common): pancreatic cancer (~90%), colorectal cancer (~40%), non-small cell lung cancer (~30%)
• Mutations are typically at codon 12 or 13 - these impair GTPase activity, locking Ras in the GTP-bound (active) state
• NF-1 (neurofibromatosis type 1): loss of neurofibromin, a Ras-GAP -> uncontrolled Ras activity -> café-au-lait spots, neurofibromas, higher cancer risk
• HRAS mutations: Costello syndrome
• KRAS/NRAS mutations: Noonan syndrome (RASopathies)

Rab Family

• Small G proteins involved in membrane vesicle trafficking
• Named: "Ras-related proteins in brain"
• Modified by isoprenylation and associate with specific membrane compartments
• Unlike Ras, GTP vs GDP binding to Rab regulates its association with specific membrane compartments (not just activity)
• Rab3 is specifically implicated in exocytosis and neurotransmitter release at nerve terminals

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13. Ion Channel Regulation via G Proteins

GIRK Channels (G protein-coupled Inwardly Rectifying K+ channels)

• Opened by free βγ subunits (not Gα - this was the historical controversy)
• Coupled to pertussis toxin-sensitive G proteins (Gi/Go family)
• Receptors that open GIRK:
  • Opioid receptors (mu, delta, kappa)
  • Alpha-2 adrenergic
  • D2 dopamine
  • M2 muscarinic
  • 5-HT1A serotonin
  • GABA-B

Voltage-Gated Ca2+ Channels

• Inhibited by these same Gi/Go-coupled receptors (same receptor list as above)
• Mechanism: free βγ directly inhibits the Ca2+ channel
• Effect: reduced Ca2+ entry -> reduced neurotransmitter release (presynaptic) or reduced contractility

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14. Membrane Trafficking Roles of Gαi

• Gαi has been detected at high concentrations in intracellular membranes: Golgi complex, trans-Golgi network, ER
• May regulate budding of membrane vesicles from these organelles
• May be involved in endocytosis (internalization of plasma membrane into cytoplasm)
• This extends the function of G proteins beyond GPCR signaling to general membrane biology

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15. Summary of Key Receptor-G Protein Pairings (USMLE High Yield Table)

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16. Classic USMLE Vignette Triggers

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17. Key Mnemonics Summary

• "Gs = Go (accelerate), Gi = Stop" - for cAMP
• "Gq = PLC-beta = Ca2+ and PKC" - like a "question" (Gq) that releases two second messengers (IP3 and DAG)
• "FLAT-DQ" - Gq receptors: FSH (no - FSH is Gs), actually: Alpha-1, M1/M3, H1, V1 (vasopressin), Oxytocin, Angiotensin II (AT1) use Gq
• "Gs BAD" = Beta adrenergic, Adenylyl cyclase activated, D1/D5 dopamine
• GTPase = OFF switch - anything inhibiting GTPase (cholera toxin, Ras mutation, RGS protein loss) locks system ON
• RGS = "Really Good Suppressor" - turns G protein signaling OFF faster
• PLCβ vs PLCγ - β = G protein (Gq); γ = growth factor (RTK)

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