Neurochemistry Lecture: G Proteins and Cell-Surface Receptors

Cell-Surface Receptors and Transmembrane Signaling

• Cell-surface receptors employ four distinct 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 activity
     - Phosphoinositide-specific phospholipase C activity modulation

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G-Protein-Coupled Receptors (GPCRs)

• GPCRs are the most diverse numerically, operating via an intervening G protein.

• Characterized by a seven transmembrane domain (7-TMD) structure.

• Divided into four functional categories:1. Regulation of K+ conductance independently of second messenger production- Examples: GABA B, α2-adrenergic, D2-dopaminergic, M2 muscarinic (mAChR)

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  2. Modulation of adenylyl cyclase activity- Positive regulation: β2-adrenergic receptor activation.

     • Negative regulation: α2-adrenergic receptor activation.

     • Changes in cAMP concentrations regulate protein kinase A (PKA) activity.

     • Example: M2 mAChRs regulate both K+ conductance and adenylyl cyclase activity in the heart.

  3. Activation of phosphoinositide-specific phospholipase C (PLC)- Breakdown of PIP2 (phosphatidylinositol 4,5-bisphosphate) leads to the formation of IP3 (inositol triphosphate) and DAG (diacylglycerol).

     • Linked to changes in Ca2+ homeostasis and protein phosphorylation via protein kinase C (PKC).

     • Other effector enzymes regulated by IP3-linked GPCRs: phospholipases A2 and D.

  4. Activation via light (rhodopsin)- Rhodopsin is a prototypical GPCR activated by light.

     • Chromophore: 11-cis-retinal, covalently bound to opsin.

     • Upon light absorption, 11-cis-retinal isomerizes to all-trans-retinal.

     • Photoactivated rhodopsin activates transducin (a G-protein).

     • Transducin is coupled to cGMP phosphodiesterase, increasing the hydrolysis rate of cGMP to GMP.

     • Loss of cGMP results in the closure of plasma membrane cation channels in rod outer segments.

G Proteins

• Diverse family of cellular proteins with various functions.

• Bind guanine nucleotides: guanosine triphosphate (GTP) and guanosine diphosphate (GDP).

• Possess intrinsic GTPase activity.

• Central role in signal transduction and cellular processes, including membrane vesicle transport, cytoskeletal assembly, cell growth, and protein synthesis.

• 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 the activation of plasmalemma receptors to intracellular processes.

• Most neurotransmitter, peptide hormone, cytokine, and chemokine receptors are GPCRs.

• Influence numerous effector proteins:- Ion channels

  • Adenylyl cyclase

  • Phosphodiesterase (PDE)

  • Phosphoinositide-specific phospholipase C (PI-PLC): Catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2).
    

    $PIP2 \rightarrow IP3 + DAG$

  • 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):- Inhibits adenylyl cyclase.

    • Activates certain K+ channels.

    • Inhibits voltage-gated Ca2+ channels.

    • Activates the MAP-kinase pathway.

    • Activates phosphodiesterase.

  • Gq family: Activates PI-PLC.

  • G12 family (G11-16): Activates Rho-GEFs (guanine nucleotide exchange factors).

• Functional activity specificity conferred by distinct α subunits.

• Multiple subtypes of β (5 subunits, Mr 35,000–36,000) and γ (11 subunits, Mr 6,000–9,000) subunits exist.- Distinct cellular distributions and functional properties.

  • β subunits divided into two families: Gβ1–4 and Gβ5.

  • Some γ subunits show striking regional distributions (e.g., Gγ7 enriched in striatum).

• All G protein α subunits are modified in their N-terminal domains by palmitoylation or myristoylation.- Regulates affinity for βγ subunits.

  • Determines association with the plasma membrane or diffusion into the cytoplasm.

• 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 to the receptor causes a conformational change in the receptor, triggering a conformational change in the α subunit.

• Decreased affinity of the α subunit for GDP, leading to GDP dissociation and GTP binding (GTP concentration is higher than GDP).

• Dissociation of a βγ subunit dimer from the α subunit.

• Release of the G protein from the receptor.

Functional Cycle of Heterotrimeric G Proteins

• Basal state: G protein exists as a heterotrimer (α, β, γ) with GDP bound to the α subunit, loosely associated with the receptor.

• Receptor Activation: Ligand binding causes the receptor to associate with the α subunit, leading to GDP dissociation and GTP binding.

• Subunit Dissociation: GTP binding causes α subunit dissociation from βγ subunits and the receptor, resulting in free α-GTP and βγ dimers.- Free α-GTP and βγ dimers regulate effector proteins (ion channels, adenylyl cyclase, phospholipase C, phospholipase A2, phosphodiesterase).

• Deactivation: Intrinsic GTPase activity of the α subunit hydrolyzes GTP to GDP, causing reassociation of α and βγ subunits, ligand dissociation, and restoration of the basal state.

G Protein Subunits and Ion Channel Gating

• G protein subunits can directly gate specific ion channels.

• Example: Coupling of receptors to inward-rectifying K+ channels (GIRK) via pertussis toxin-sensitive G proteins (Gi/o subtypes) in neurons.

• Coupled receptors: Opioid, α2-adrenergic, D2-dopaminergic, muscarinic cholinergic, 5-HT1A-serotonergic, and GABA-B receptors.

• βγ complex is primarily responsible for this action.

• The same receptors are coupled via pertussis toxin-sensitive G proteins to voltage-gated Ca2+ channels, inhibiting them.

G Proteins and Intracellular cAMP Concentrations

• G proteins control cAMP concentrations by mediating neurotransmitters' ability to activate or inhibit adenylyl cyclase.

• Activation of receptors coupled to Gs results in free Gαs subunits, directly activating adenylyl cyclase.

• Free βγ-subunit complexes also activate certain subtypes of adenylyl cyclase.

• Gαolf, structurally related to Gαs and enriched in olfactory epithelium and striatum, follows a similar mechanism.

• The transducin family regulates phosphodiesterase, affecting cyclic nucleotides (cAMP, cGMP) in the visual system.

• Gαt activates PDE via direct binding.

• Gustducin (Gαgust), enriched in taste epithelium, mediates signal transduction via a distinct phosphodiesterase.

G Proteins and the Phosphoinositide Second Messenger Pathway

• Stimulation of the phosphoinositide pathway is mediated by PLC activation, which hydrolyzes PIP2 to form IP3 and DAG.

• Receptor-induced PLC activation is mediated via G proteins, specifically regulating the β isoform of PLC (PLCβ).

• The γ isoform of PI-PLC is regulated by growth factors and tyrosine kinase receptors.

• Gq mediates neurotransmitter regulation of PLCβ; Gαq and related α subunits bind to and activate the enzyme directly.

• In some cells, βγ subunits from Gi proteins directly bind and activate PLCβ.

G Proteins and Membrane Trafficking

• Heterotrimeric G proteins are implicated in membrane trafficking processes.

• Gαi subunit is detected in intracellular membranes, including the Golgi complex, trans-Golgi network, and endoplasmic reticulum.

• Gαi may regulate the budding of membrane vesicles.

• Involved in the vesicularization of portions of the plasma membrane into the cytoplasm via endocytosis.

G Protein-Receptor Kinases (GRKs) and Other Binding Proteins

• GRKs phosphorylate ligand-occupied GPCRs, mediating receptor desensitization.

• βγ subunits bind to GRKs, certain protein kinases, phosducin, and Ras-GEFs.

• Phosducin: phosphoprotein in rod cells of the retina.

Role of G Protein βγ Subunits in Intracellular Targeting of Proteins

• Resting Conditions: The receptor is loosely associated with a heterotrimeric G protein, and GRKs are cytoplasmic.

• Activation: Receptor and G protein activation generate free α subunits (leading to physiological effects) and βγ dimers.

• βγ dimers bind to GRKs, drawing them to the membrane to phosphorylate ligand-occupied receptors, directing GRKs to their targets.

• The βγ complex is tethered to the membrane by an isoprenyl group on the γ subunit.

• Free βγ also produces other physiological effects by interacting with other cellular proteins.

G Protein βγ Subunits and the MAP-Kinase Pathway

• βγ subunits regulate the mitogen-activated protein kinase (MAP-kinase) pathway, including ERK (extracellular-regulated kinase).

• Signals through GPCRs (particularly those coupled to Gi) can modulate growth factor activation of the MAP-kinase pathway, mediated via βγ subunits.

• Receptor activation leads to free βγ subunits, which activate the MAP-kinase pathway early in the cascade.

• Possible mechanisms include direct action on Ras or 'linker' proteins.

• Gβγ subunits can mediate G-protein signaling without receptor activation via the GoLoco protein, which triggers the release of free βγ dimers.

Phosducin Modulation of G Protein βγ Subunits

• Phosducin, a cytosolic protein enriched in the retina and pineal gland, binds to G protein βγ subunits with high affinity.

• Prevents βγ subunit reassociation with the α subunit.

• May sequester βγ subunits, prolonging the α subunit's activity but eventually inhibiting G protein activity by preventing the direct effects of βγ and regeneration of the heterotrimer.

• The ability of phosducin to bind βγ is altered upon phosphorylation by cAMP- or Ca2+-dependent protein kinases.

Regulators of G Protein Signaling (RGS) Proteins

• GTPase-activating proteins (GAPs) modulate the activity of small G proteins by stimulating GTPase activity.

• RGS proteins, analogous proteins for heterotrimeric G proteins, bind to G protein α subunits and stimulate their GTPase activity.

• Hastens the hydrolysis of GTP to GDP, 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, and other domains that control localization, stability, and other functions.

• Alterations in RGS protein activity modulate the activity of specific G proteins and, consequently, the sensitivity of specific GPCRs.

• Implicated in disorders such as hypertension, drug addiction, schizophrenia, and Parkinson’s disease.

RGS12 Subfamily and Go-Loco Motif

• R12 subfamily of RGS proteins contains the Go-Loco motif.

• Go-Loco binds directly to Gi and stabilizes it in its GDP-bound form, leading to the dissociation of Gβγ subunit dimers, independently of receptor activation.

• RGS12 proteins stimulate receptor-independent G protein signaling.

Activators of G Protein Signaling (AGS)

• AGS are structurally diverse proteins that serve as binding partners of G protein components independent of receptor activation.

• G proteins, receptors, and RGS proteins can undergo phosphorylation by protein serine/threonine kinases and protein tyrosine kinases.

Small G Proteins

• Like heterotrimeric G proteins, small G proteins bind guanine nucleotides, possess intrinsic GTPase activity, and cycle through GDP- and GTP-bound forms.

• Function as molecular switches controlling cellular processes.

• The best-characterized small G protein is the Ras family (21 kDa).

Ras Proteins

• Identified originally as oncogene products of Harvey and Kirsten rat sarcoma viruses.

• Normal cellular homologs (proto-oncogenes) of viral Ras were identified.

• Mammalian Ras proteins are encoded by three genes: H-ras, K-ras, and N-ras, found in diverse mammalian tissues, including the brain.

• All three forms are membrane-associated proteins.

• Ras activity is highly regulated by associated proteins:- Guanine nucleotide exchange factors (GEFs) stimulate the release of GDP, facilitating GTP binding.

  • GAPs bind to Ras and activate its GTPase activity, reducing functional activity.

  • GTPase-inhibitory proteins (GIPs) inhibit GTPase activity.

  • Guanine nucleotide dissociation inhibitors (GDIs) reduce the rate of GDP exchange for GTP.

Regulation of G Protein Function

• The functional activity of G proteins is controlled by cycles of binding GDP versus GTP, associated with conformational changes.

• There are proteins that facilitate GDP release (GEFs) and GTPase activation (GAPs).

• Heterotrimeric βγ subunits and analogous proteins for small G proteins may act as GTPase inhibitory proteins (GIPs).

• Phosducin modulates G protein function by binding to βγ subunits.

Ras and MAP-Kinase Pathways

• Numerous cell signals, including growth factors, converge on Ras to regulate MAP-kinase pathways.

• Activation of growth factor receptors results in GEF activation, termed Sos, which activates Ras.

• Activated Ras binds to the N-terminal domain of Raf, the first protein kinase in the MAP-kinase pathway.

• Ras appears to activate Raf indirectly.

• Anchoring of Ras in the plasmalemma may be mediated by isoprenylation.

Rab Proteins

• Rab is a family of small G proteins involved in membrane vesicle trafficking.

• Rab proteins (ras-related proteins in brain) are isoprenylated and associate with membranes.

• GTP and GDP binding to Rab regulate its association with membrane compartments.

• Subtypes of Rab, particularly Rab3, are implicated in the regulation of exocytosis and neurotransmitter release at nerve terminals.

MAP-Kinase Cascade Mediated by Ras

• Activation of growth factor receptors like EGF receptors by ligands such as EGF initiates a signaling cascade.

• Adaptor proteins such as Grb2 bind to the activated receptor, recruiting GEFs like SOS.

• SOS stimulates the exchange of GDP for GTP on Ras, activating it (Ras-GTP).

• Activated Ras recruits and activates Raf-1, a serine/threonine kinase.

• Raf-1 phosphorylates and activates MEK (MAPK/ERK kinase).

• MEK phosphorylates and activates ERK (extracellular signal-regulated kinase).

• ERK translocates to the nucleus and phosphorylates transcription factors like Elk-1 and SRF.

• Phosphorylated Elk-1 and SRF regulate the transcription of target genes, leading to cellular responses.