Neurochemistry Notes: G Proteins

Cell-Surface Receptors and Transmembrane Signaling

• Cell-surface receptors use four molecular mechanisms for transmembrane signaling:
  1. Ligand-gated ion channels
  2. Receptors with intrinsic guanylyl cyclase activity
  3. Receptors with intrinsic or associated tyrosine kinase activity
  4. G-protein-coupled receptors (GPCRs) linked to:
     • Opening/closing of ion channels
     • Modulation of adenylyl cyclase
     • Phosphoinositide-specific phospholipase C (PLC) activities

G Protein-Coupled Receptors (GPCRs)

• GPCRs are the most diverse type of receptor operating via an intervening G protein.
• They have a characteristic seven transmembrane domain (7-TMD) structure.
• GPCRs can be divided into four functional categories:
  1. Regulation of K+ conductance independently of second messengers:
     • Examples: GABA B, α2-adrenergic, D2-dopaminergic, and M2 muscarinic (mAChR) receptors.
  2. Modulation of adenylyl cyclase activity:
     • Positive regulation: Activation of β2-adrenergic receptor (increases cAMP).
     • Negative regulation: Activation of α2-adrenergic receptor (decreases cAMP).
     • Changes in cAMP concentrations regulate protein kinase A (PKA) activity.
  3. Activation of phosphoinositide-specific phospholipase C (PLC):
     • Leads to the breakdown of $PIP<em>2$ and formation of $IP</em>3$ and DAG.
     • Affects $Ca^{2+}$ homeostasis and protein phosphorylation via protein kinase C (PKC).
     • May also regulate phospholipases A2 and D.
  4. Unique mechanism: Rhodopsin activation:
     • Visual pigment rhodopsin is a prototypical GPCR.
     • Activated by light instead of a chemical stimulus.
     • The chromophore, 11-cis-retinal, isomerizes to all-trans-retinal upon light absorption.
     • Photoactivated rhodopsin activates transducin (a G-protein).
     • Transducin is coupled to cGMP phosphodiesterase, increasing the hydrolysis of cGMP to GMP.
     • Loss of cGMP results in the closure of plasma membrane cation channels in rod outer segments.

G Proteins

• G proteins are a diverse family of cellular proteins with varied functions.
• They bind guanine nucleotides: guanosine triphosphate (GTP) and guanosine diphosphate (GDP).
• They possess intrinsic GTPase activity.
• They play a central role in signal transduction and cellular processes like:
  • Membrane vesicle transport
  • Cytoskeletal assembly
  • Cell growth
  • Protein synthesis
• Mammalian G proteins are divided into two major categories:
  • Heterotrimeric G proteins
  • Small G proteins

Heterotrimeric G Proteins

• Involved in transmembrane signaling in the nervous system (with exceptions).
• Consist of three distinct subunits: α, β, and γ.
• Couple activation of plasmalemma receptors to intracellular processes.
• Most neurotransmitter, peptide hormone, cytokine, and chemokine receptors are GPCRs.
• Influence numerous effector proteins, including:
  • Ion channels
  • Adenylyl cyclase
  • Phosphodiesterase (PDE)
  • Phosphoinositide-specific phospholipase C (PI-PLC): Catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate ($PIP_2$).
  • Phospholipase A2 (PLA2): Catalyzes the hydrolysis of membrane phospholipids to yield arachidonic acid.
• Implicated in vesicular transport and cytoskeletal assembly.

Types of G Proteins

• Divided into four main categories:
  • Gs family: Stimulates adenylyl cyclase.
  • Gi family: Includes Go, Ggust, Gz. Can:
    • Inhibit adenylyl cyclase
    • Activate a certain type of $K^+$ channel
    • Inhibit voltage-gated $Ca^{2+}$ channels
    • Activate the MAP-kinase pathway
    • Activate phosphodiesterase
  • Gq family: Activates PI-PLC.
  • G12 family: Composed of G11-16. Activates Rho-GEFs (guanine nucleotide exchange factors).
• Different types of G protein contain distinct α subunits, which confer functional activity specificity.

G Protein Subunits

• Multiple subtypes of β and γ subunits exist:
  • Five β subunits: $M_r$ 35,000–36,000
  • Eleven γ subunits: $M_r$ 6,000–9,000
  • Show distinct cellular distributions and functional properties.
  • β subunits are divided into two families: Gβ1–4 and Gβ5.
  • γ subunits are more structurally divergent; some show striking regional distributions in the brain (e.g., Gγ7 in striatum).
• All G protein α subunits are modified in their N-terminal domains by palmitoylation or myristoylation.
  • These modifications may regulate the affinity of the α subunit for its βγ subunits.
  • They may also determine whether the α subunit remains associated with the plasma membrane or diffuses.
• G protein γ subunits are modified on their C-terminal cysteine residues by isoprenylation.

G Protein Activation Mechanism

• In the resting state, G proteins exist as heterotrimers that bind GDP and are associated with extracellular receptors.
• Ligand binding activates the receptor, causing a conformational change.
• This change triggers a conformational change in the α subunit of the G protein.
• The α subunit's affinity for GDP decreases, leading to GDP dissociation and GTP binding (due to higher GTP concentration).
• A βγ subunit dimer dissociates from the α subunit.
• The G protein is released from the receptor.

Functional Cycle of Heterotrimeric G Proteins

• (A) Basal Conditions: G proteins exist as heterotrimers loosely associated with neurotransmitter receptors; GDP is bound to the α subunit.
• (B) Receptor Activation: Ligand binding causes the receptor to associate with the α subunit, leading to GDP dissociation and GTP binding.
• (C) GTP Binding: Induces the generation of free α subunits (bound to GTP) by dissociation from β and γ subunits and the receptor.
  • Free α subunits (GTP-bound) and free βγ subunit dimers are functionally active.
  • They regulate effector proteins like ion channels, adenylyl cyclase, phospholipase C, phospholipase A2, and phosphodiesterase.
• (D) GTP Hydrolysis: Intrinsic GTPase activity of the α subunit degrades GTP to GDP.
  • This leads to the reassociation of α and βγ subunits and ligand dissociation from the receptor, restoring the basal state.

G Protein Subunits and Ion Channels

• G protein subunits can directly gate specific ion channels.
• Example: Coupling of receptors to the activation of inward-rectifying $K^+$ channels (GIRK) via pertussis toxin-sensitive G proteins (Gi/o) in neurons.
  • Coupled receptors include opioid, α2-adrenergic, D2-dopaminergic, muscarinic cholinergic, 5-HT1A-serotonergic, and GABA-B receptors.
• The βγ complex is primarily responsible for this action.
• These neurotransmitter receptors are also coupled via pertussis toxin-sensitive G proteins to voltage-gated $Ca^{2+}$ channels.
  • Channels are inhibited by the interaction.

G Proteins and Intracellular cAMP Concentrations

• G proteins control intracellular cAMP concentrations by mediating the ability of neurotransmitters to activate or inhibit adenylyl cyclase.
• Activation of neurotransmitter receptors coupled to Gs results in the generation of free Gαs subunits, which directly activate adenylyl cyclase.
• Free βγ-subunit complexes can also activate certain subtypes of adenylyl cyclase.
• Gαolf, related to Gαs, is enriched in olfactory epithelium and striatum.
• The transducin family mediates signal transduction in the visual system by regulating specific forms of phosphodiesterase that catalyze cyclic nucleotide metabolism (cAMP, cGMP).
• Gαt activates PDE via direct binding to the enzyme.
• Gustducin (Gαgust) is enriched in taste epithelium and mediates signal transduction in this tissue via a distinct form of phosphodiesterase.

G Proteins and the Phosphoinositide Second Messenger Pathway

• Many neurotransmitters stimulate the phosphoinositide pathway via PLC activation.
• PLC catalyzes the hydrolysis of $PIP<em>2$ to form inositol triphosphate ($IP</em>3$) and diacylglycerol (DAG).
• Receptor-induced activation of PLC is mediated via G proteins, specifically the β isoform of PLC.
• The γ isoform of PI-PLC is regulated by growth factors and their tyrosine kinase receptors.
• Gq mediates neurotransmitter regulation of PLCβ.
• Gαq and related α subunits bind to and directly activate the enzyme.
• In some cell types, βγ subunits released from Gi proteins directly bind and activate PLCβ.

Other Functions of G Proteins

• Heterotrimeric G proteins are implicated in membrane trafficking processes.
• Gαi subunit is detected in intracellular membranes, like the Golgi complex, trans-Golgi network, and ER.
• Gαi may regulate the budding of membrane vesicles through these organelles and may be involved in endocytosis.
• The βγ subunits also bind to several other proteins, including certain protein kinases as well as phosducin and Ras-GEFs.
  • One class of protein kinase that binds βγ subunits is called G-protein– receptor kinases (GRKs). These kinases phosphorylate G-protein–coupled receptors that are occupied by ligand and thereby mediate one form of receptor desensitization.

Role of G Protein βγ Subunits

• Intracellular Targeting of Proteins:
  • (A) Resting Conditions: Receptor is loosely associated with a heterotrimeric G protein and cytoplasmic GRKs.
  • (B) Activation: Receptor and G protein activation generates a free α subunit and a free βγ subunit dimer.
  • The free βγ subunit can bind to the GRK and draw it toward the membrane to phosphorylate the ligand-occupied receptor, directing GRKs to their targets.
  • Free βγ also produces other physiological effects by interacting with other cellular proteins.
• Regulation of the MAP-Kinase Pathway:
  • MAP-kinases are the major effector pathway for growth factor receptors.
  • G protein–coupled receptors, coupled to Gi, can modulate growth factor activation of the MAP-kinase pathway via βγ subunits.
  • Activation of the receptors leads to the generation of free βγ subunits, which then activate the MAP-kinase pathway at some early step in the cascade.
  • Some possibilities include direct action of the βγ subunits on Ras or on one of several ‘linker’ proteins between the growth factor receptor itself and activation of Ras.
• Gβγ subunits in mediating G-protein signalling in the absence of activation of the G protein’s associated receptor:
  * Involves a newly discovered modulatory protein, called GoLoco, which triggers the release of free βγ dimers from G protein–receptor complexes without receptor activation and leads to βγ regulation of its several effector proteins.

Modulators of G Protein βγ Subunit Activity

• The activity of G protein βγ subunits is modulated by phosducin.
  • Phosducin: A cytosolic protein enriched in retina and pineal gland but also expressed in brain and other tissues.
  • It binds to G protein βγ subunits with high affinity, preventing their reassociation with the α subunit.
  • This may initially prolong the biological activity of the α subunit.
  • Eventually, it may inhibit G protein activity by preventing the direct biological effects of βγ subunits and preventing regeneration of the functional heterotrimer.
  • The ability of phosducin to bind to βγ subunits is altered upon its phosphorylation by cAMP- or $Ca^{2+}$ -dependent protein kinases.

Regulation of Heterotrimeric G Proteins

• GTPase-Activating Proteins (GAPs): Stimulate GTPase activity.
  • RGS proteins bind to G protein α subunits and stimulate their GTPase activity, hastening GTP hydrolysis to GDP and restoring the inactive heterotrimer.
  • RGS proteins inhibit the biological activity of G proteins.
  • All RGS proteins contain a core RGS domain responsible for regulating GTPase activity, while other domains may control localization, stability, and other functions.
  • Alterations in RGS protein activity can modulate the activity of specific G proteins and the sensitivity of specific GPCRs, implicated in disorders like hypertension, drug addiction, schizophrenia, and Parkinson’s disease.
• Go-Loco Motif: Present in the R12 subfamily of RGS proteins.
  • Binds directly to Gi and stabilizes it in its GDP-bound form, leading to dissociation of Gβγ subunit dimers that activate effectors.
  • Stimulates receptor-independent G protein signaling.
• Activators of G Protein Signaling (AGS): Structurally diverse proteins that bind to G protein components independently of receptor activation.
• Phosphorylation: G proteins, associated receptors, and RGS proteins can undergo phosphorylation by serine/threonine kinases and tyrosine kinases.

Small G Proteins

• Like heterotrimeric G proteins, they bind guanine nucleotides, possess intrinsic GTPase activity, and cycle through GDP- and GTP-bound forms.
• They function as molecular switches that control several cellular processes.
• The best-characterized family is the Ras family (21 kDa proteins).

Ras Proteins

• Originally identified as oncogene products of Harvey and Kirsten rat sarcoma viruses.
• Normal cellular homologs (proto-oncogenes) of viral Ras were subsequently identified.
• Mammalian Ras proteins are encoded by three homologous genes:
  • Proto-oncogene for Harvey Ras virus (H-ras)
  • Proto-oncogene for Kirsten Ras virus (K-ras)
  • Neural Ras (N-ras)
• All three forms are found in diverse mammalian tissues, including the brain.
• All three forms of Ras are membrane-associated proteins.

Regulation of Ras Activity

• Regulated by several associated proteins:
  • Guanine Nucleotide Exchange Factors (GEFs):
    • Stimulate the release of GDP from inactive Ras, facilitating GTP binding.
    • Increase Ras activity.
  • GTPase-Activating Proteins (GAPs):
    • Bind to Ras and activate its intrinsic GTPase activity.
    • Reduce Ras functional activity.
  • GTPase-Inhibitory Proteins (GIPs):
    • Bind Ras and inhibit GTPase activity.
  • Guanine Nucleotide Dissociation Inhibitors (GDIs):
    • Reduce the rate of GDP exchange for GTP.

Modulators of G Protein Function

• The functional activity of G proteins is controlled by cycles of binding GDP versus GTP, associated with a major conformational change.
• Several proteins regulate this cycle:
  • GEFs: Facilitate GDP release and enhance G protein function.
    • Examples: Receptors for heterotrimeric G proteins or GEFs specific for small G proteins.
  • GAPs: Activate GTPase activity and inhibit G protein function.
    • Examples: RGS proteins for heterotrimeric G proteins and GAPs specific for small G proteins.
  • GIPs: Exert the opposite effects of GAPs
  • Phosducin binds to βγ subunits, representing another regulatory protein that modulates G protein function.

Ras and MAP-Kinase Pathways

• Numerous cell signals, including many growth factors, converge on Ras to regulate MAP-kinase pathways.
• Activation of growth factor receptors results in the activation of a GEF, termed Sos, which activates Ras.
• Activated Ras then binds to the N-terminal domain of a protein kinase called Raf, the first protein kinase in the MAP-kinase pathway.
• Ras appears to activate Raf via an indirect mechanism.
• Anchoring of Ras in the plasmalemma may be mediated by isoprenylation.

Rab Proteins

• A family of small G proteins involved in membrane vesicle trafficking vesicles and organelles that exist in cells.
• Rab proteins, named originally as ras-related proteins in the brain, are isoprenylated and associate with membranes, as do isoprenylated Ras and G protein γ subunits.
• GTP and GDP binding to Rab appear to regulate its association with membrane compartments.
• Subtypes of Rab, particularly Rab3, have been implicated in the regulation of exocytosis and neurotransmitter release at nerve terminals.