Lec 21 GPCR Signaling and Calcium Messengers

GPCRs and their Second Messengers (Section 15.3)

Receptors can be classified into basic categories:
- Ligand-gated channels
- Plasma membrane receptors
- Those linked to G proteins
- Those linked to protein kinases
- Nuclear receptors
- G Protein-Coupled Receptors (GPCRs) are named for their interaction with heterotrimeric G proteins (guanine-nucleotide binding proteins).
- GPCRs constitute the largest superfamily in the human genome and characteristically have $7$ transmembrane α-helices.
- The extracellular portion contains a unique ligand-binding site; the cytosolic portion interacts with specific G proteins.
GPCR structure and binding
- GPCRs have $7$ transmembrane α-helices with a ligand-binding pocket formed by the three extracellular loops (these loops vary among GPCRs).
- The three intracellular loops provide binding sites for signaling proteins, including heterotrimeric G proteins.
- Inactive state is stabilized by noncovalent interactions within the transmembrane α-helices that bury the G-protein binding sites; ligand binding disrupts these interactions, rotates the helices, and unmasks G-protein binding sites.
Activation cycle (overall pathway)
1) Ligand binding to the GPCR changes receptor conformation, unmasks the G protein binding site, and increases GPCR–G protein affinity.
2) The Ga subunit exchanges GDP for GTP, activating Ga; in the GTP-bound state, Ga has a reduced affinity for the Gβγ dimer, leading to dissociation of the heterotrimeric complex.
- ext{Ga-GDP}
 ightarrow ext{Ga-GTP} igg[ ext{activation via exchange (GEF activity of GPCR)}igg]
 3) Ga-GTP (active) interacts with an effector protein (e.g., adenylyl cyclase) to propagate the signal. One active GPCR can activate multiple G proteins (GPCR serves as a GEF).
 4) Activation of the effector leads to production of a second messenger (e.g., $ext{cAMP}$ for adenylyl cyclase pathway) that activates downstream signaling proteins.
 5) The Ga subunit hydrolyzes GTP to GDP and Pi, returning to the inactive state and increasing its affinity for Gβγ, promoting reformation of the heterotrimeric complex.
- $ext{Ga-GTP} ightarrow ext{Ga-GDP} + ext{Pi}$
 6) Desensitization prevents overstimulation: GPCR kinases (GRKs) phosphorylate the cytoplasmic domain; arrestins bind and compete with G proteins for receptor binding.
 7) Arrestin binding can target the receptor to clathrin-coated pits via interaction with AP2, leading to receptor endocytosis.
- Outcomes after endocytosis:
 - Re-sensitization: receptor may return to the surface and resume signaling.
 - Endosomal signaling: receptor can continue signaling within endosomes.
 - Degradation: receptor can be trafficked to lysosomes, reducing ligand sensitivity.
Key features of GPCR signaling
- GPCR signaling can couple to multiple G proteins and effectors; the same receptor can activate more than one G protein.
- GPCRs can be stimulatory (Gs type) or inhibitory (Gi type) depending on the Ga subunit.
- The Ga subunit binds GDP/GTP and cycles between active (Ga-GTP) and inactive (Ga-GDP) states; Gβγ can also signal independently.
- The Gβγ subunits: Geta and $G ext{gamma}$ can modulate other effectors (e.g., activate K+ channels) after dissociation.
- There are two classes of G proteins:
- small monomeric G proteins (e.g., Ran, Rab, Rho, Ras) — $ext{monomeric}$
- large heterotrimeric G proteins — $ext{Ga}$ , Geta, $G ext{gamma}$
G protein subunit diversity and membrane anchoring
- Heterotrimeric G proteins are anchored to the plasma membrane via lipid modifications:
- Ga via fatty acid anchoring
- Gβγ via isoprenylation and related lipid links
- The human genome encodes multiple subunits: $16$ different $G_ ext{α}$ subunits, $5$ different Geta subunits, and $11$ different $G ext{γ}$ subunits.
Core second messengers and downstream cascades
- Common second messengers regulated by GPCRs include $cAMP$ , $IP_3$ , $DAG$ , and $Ca^{2+}$ .
- GPCRs that activate or inhibit adenylyl cyclase influence $cAMP$ levels; PDEs (phosphodiesterases) hydrolyze $cAMP$ to $5'-AMP$ , thus terminating the signal.
- Key example: adenylyl cyclase converts $ATP$ to $cAMP$ and $PP_i$ (pyrophosphate) as a byproduct:
- $ext{ATP} ightarrow ext{cAMP} + ext{PP}_{i}$
- $cAMP$ activates Protein Kinase A (PKA), which phosphorylates various substrates on serine/threonine residues, using ATP as the phosphate donor.
- The glycogen pathway illustrates downstream effects: cAMP-activated PKA promotes glycogen breakdown and inhibits glycogen synthesis through phosphorylation of specific enzymes.
cAMP signaling and organization of substrates
- Different cell types respond differently to increased $cAMP$ due to variation in substrates and scaffold proteins.
- AKAPs (A Kinase-Anchoring Proteins) scaffold PKA to specific cellular locations, aligning it with nearby substrates and creating spatially restricted signaling hubs.
- As a result, the same second messenger (cAMP) can elicit distinct outcomes in different cell types depending on AKAP composition and substrate availability.
Phospholipase C–β pathway (Gq signaling)
- GPCRs can couple to $Gq$ (or $G{aq}$ ) leading to activation of phospholipase C-β (PLC-β).
- PLC-β cleaves PIP2, a membrane lipid, to yield two second messengers: $IP_3$ and DAG:
- $ext{PIP}2 ightarrow ext{IP}3 + ext{DAG}$
- IP3 is soluble and diffuses to bind IP3 receptors on the ER, triggering Ca^{2+} release into the cytosol.
- DAG remains in the membrane and helps activate Protein Kinase C (PKC).
Calcium (Ca^{2+}) as a versatile second messenger
- Cytosolic Ca^{2+} is normally kept at low levels; high levels trigger diverse cellular responses.
- Maintenance mechanisms keep cytosolic Ca^{2+} low: Ca^{2+} channels in the ER and plasma membrane are kept closed; Ca^{2+} ATPases and Ca^{2+} exchangers pump Ca^{2+} out of the cytosol.
- Signaling raises cytosolic Ca^{2+} by:
- Opening voltage-gated Ca^{2+} channels in response to nerve impulses.
- Opening IP3-gated Ca^{2+} release channels (IP3 receptor channels).
- Ryanodine receptor channels (Ca^{2+-induced Ca^{2+}} release) respond to cytosolic Ca^{2+} levels.
- Calcium signaling is often amplified through Ca^{2+}-binding proteins and downstream enzymes.
Ca^{2+}-binding proteins and effectors
- A major calcium-binding protein is calmodulin (CaM): a small (~ $17$ kDa) protein that binds $4$ Ca^{2+} ions cooperatively and undergoes a conformational change upon Ca^{2+} binding.
- Calcium-bound CaM activates numerous targets, including:
- Ca^{2+}/calmodulin-dependent kinases (CaMK)
- Myosin light chain kinase (MLCK)
- Calcineurin (a phosphatase)
- Various ion channels
- Calmodulin serves as a central mediator translating Ca^{2+} signals into phosphorylation/dephosphorylation events and other responses.
Ca^{2+}-dependent signaling and cytoskeletal/motor effects
- Ca^{2+}-dependent kinases and phosphatases regulate motor proteins and cytoskeletal elements, contributing to processes such as muscle contraction, vesicle trafficking, and secretion.
The role of Ca^{2+} signaling in conjunction with other messengers
- Calcium signaling often intersects with cAMP/PKA signaling and PKC pathways, creating integrated networks that regulate diverse cellular processes.
- The same Ca^{2+} signal can have different outcomes depending on the spatial localization (e.g., near AKAPs or substrates) and the complement of Ca^{2+}-binding proteins present.
Experimental visualization of Ca^{2+} dynamics
- Ca^{2+} dynamics can be visualized in living cells using Ca^{2+}-sensitive dyes (e.g., fura-2) that fluoresce upon binding Ca^{2+}; increases in cytosolic Ca^{2+} can be tracked in real time.
- Example: adrenocortical cells stained with fura-2 show low Ca^{2+} in unstimulated cells and elevated Ca^{2+} after GPCR activation and PLC-β–mediated IP3 generation leading to Ca^{2+} release from stores.
Key takeaways and connections
- GPCR activation triggers second messenger cascades (e.g., $cAMP$ , $IP_3$ , DAG, $Ca^{2+}$ ) that propagate signals to multiple downstream targets.
- Signaling fidelity is achieved via spatial organization (AKAPs), temporal control (phosphorylation/dephosphorylation cycles), and receptor desensitization/endocytosis mechanisms.
- Calcium acts as a central intracellular messenger, mediating numerous downstream processes through Ca^{2+}-binding proteins, with CaM as a primary mediator.
Practical implications
- GPCR signaling modulates physiological processes including vision, smell, taste, and glucose regulation; dysregulation can contribute to disease and is a major target for therapeutics (e.g., opioid receptors as clinically relevant GPCRs).
- Understanding GPCR and Ca^{2+} signaling is critical for interpreting drug actions and cellular responses in physiology and pharmacology.
Quick reference: core questions to review
- Know G-protein structure and the distinction between heterotrimeric and monomeric G proteins; recognize the roles of Ga, Gβ, and Gγ subunits and how they anchor to membranes.
- Be able to outline the GPCR activation cycle, from ligand binding to GTP/GDP exchange, effector activation, second messenger production, and signal termination.
- Understand the desensitization process via GRKs and arrestins; recognize the outcomes of receptor endocytosis.
- Distinguish between the cAMP/PKA pathway and the PLC-β/IP3/DAG pathway and how Ca^{2+} integrates into these signaling networks.

The Role of Calcium as a Messenger (Section 15.7)

Why Ca^{2+} is a key intracellular messenger
- Ca^{2+} regulates a wide variety of cellular functions and acts downstream of many GPCR- and PLC-β–mediated signals.
- Cytoplasmic Ca^{2+} is normally kept at very low concentrations; signaling increases cytosolic Ca^{2+} by opening channels and releasing Ca^{2+} from internal stores.
Mechanisms maintaining and increasing cytosolic Ca^{2+}
- Homeostasis maintains low cytosolic Ca^{2+} with:
- Closed Ca^{2+} channels in the ER and plasma membrane when at rest.
- Active transport by Ca^{2+} ATPases and Na^{+/Ca^{2+}} exchangers to remove Ca^{2+} from the cytosol.
- Elevation of cytosolic Ca^{2+} can occur via:
- Voltage-gated Ca^{2+} channels opening in response to neural activity.
- IP3 receptor–mediated Ca^{2+} release from internal stores upon IP3 binding.
- Ryanodine receptor–mediated Ca^{2+} release (calcium-induced calcium release).
IP3 and Ca^{2+} release
- IP3, generated by PLC-β cleavage of PIP2, binds IP3 receptors on the endoplasmic reticulum to release Ca^{2+} into the cytosol.
- The IP3–Ca^{2+} signaling axis links GPCR activation to rapid cytosolic Ca^{2+} increases.
Ca^{2+}-binding proteins and downstream effectors
- The major Ca^{2+}-binding protein is Calmodulin (CaM):
- Binds $4$ Ca^{2+} ions cooperatively and undergoes a conformational change upon binding.
- Activated Ca^{2+}-CaM complex engages and regulates multiple enzymes and channels.
- Key Ca^{2+}-CaM targets include:
- Ca^{2+}/calmodulin-dependent kinases (CaMK)
- Myosin light chain kinase (MLCK)
- Calcineurin (a phosphatase)
- Various ion channels
Kinases and phosphatases downstream of Ca^{2+}
- CaMKs phosphorylate numerous substrates, modulating metabolism, gene expression, and cytoskeletal dynamics.
- MLCK phosphorylates myosin light chains, promoting contractile activity in muscle and non-muscle cells.
- Calcineurin dephosphorylates specific substrates, influencing transcription factors and other signaling events.
Spatial organization and substrate specificity
- AKAPs (A Kinase-Anchoring Proteins) also influence calcium signaling by localizing PKA near Ca^{2+}-dependent substrates, creating cross-talk between cAMP and Ca^{2+} pathways and ensuring localized responses.
Ca^{2+} signaling in context
- Ca^{2+} does not act alone; it interacts with other messengers (e.g., cAMP, DAG, PKC) to produce integrated cellular outcomes.
- The same Ca^{2+} signal can have diverse effects depending on cell type, AKAP expression, CaM availability, and the array of downstream targets present.
Experimental visualization of Ca^{2+} signals
- Ca^{2+} indicators like fura-2 allow visualization of cytosolic Ca^{2+} dynamics in living cells, illustrating resting vs stimulated states and the trajectory of Ca^{2+} release and reuptake.
Practical implications and synthesis
- Calcium signaling provides a versatile platform for controlling muscle contraction, secretion, enzyme activity, and gene regulation.
- It is a central node in many signaling networks, often shaping the magnitude and duration of responses elicited by GPCRs and other receptors.