Lecture 17 Signal Transduction and G-Protein Coupled Receptors

extracellular signaling molecules

-synthesized, packaged into secretory vesicles, and secreted by specialized signaling cells within multicellular organisms

ligands

A molecule that binds specifically to a receptor site of another molecule.

exogenously derived molecules

serves as ligands for cellular receptors

signal

produces a specific response only in target cells expressing receptor proteins that bind the signal

activated receptor

-relays info to cytoplasmic proteins and other molecules

-relays message of: amplifying signal, integrate it w other signals, perform a cell response

endocrine signaling

-signaling molecules--synthesized and secreted by signaling cells

-transported thru the circulatory system

-affect distant target cells expressing the receptor

paracrine signaling

-signaling molecules secreted by a cell- affect only nearby target cells expressing the receptor

-some may bind to ECM -- released only when ECM is degraded

neuronal signaling

-signal delivered to individual cells over long distances

-examples: electrical impulse sent along axon to synapse; neurotransmitter released and diffuses across synapse to target cell

autocrine signaling

-(growth factors)- cells respond to signals they secrete

juxtacrine signaling

-signaling neighboring cells by direct contact with surface receptors
-or the signal is transmitted directly from cytoplasm of one cell through small conduits (gap junctions) into the cytoplasm of adjacent cell

signaling pathway

the series of steps from signaling molecule to response

-each protein in a pathway alters the conformation of next protein. usually by regulation of phosphorlyation state

-target proteins alter some cellular activity

-overall process is signal transduction

cell signaling systems

receptors reside either side of the cell membrane (surface receptors) or within the cells (intracellular receptors)

two main types of swtiches

-controlled by phosphorylation state and GTP binding proteins called G proteins

Kinase/phosphatase switch

-cell surface receptor signaling: involves kinase phosphorylation and phosphatase desphosphorylation to regulate target protein activity

-protein kinase: transfers terminal phosphate from ATP to specific Ser/Thr or Tyr-OH

-protein phosphatase: hydrolyzes P off protein restoring Ser/Thr or Tyr-OH

protein kinases and phosphatases

- regulated by signaling processes
- modify specific protein targets containing target motifs

GTPase Switch Proteins cycle between active and inactive forms

-signal transduction pathway on-off switches

GTPase protein superfamily (ON)

•ON/active – GTP bound

•OFF to ON –

•promoted by GEFs (guanine nucleotide exchange factors)

•GEFs catalyze dissociation of bound GDP and replacement by GTP (not phosphorylation of GDP)

GTPase protein superfamily (OFF)

•OFF/inactive – bound GDP

•ON to OFF –

•GTPase activity – GTP → GDP + Pi (ON to OFF)

•Accelerated by GAPs (GTPase-activating proteins) and RGSs (regulators of G protein signaling)

•Stay OFF/Prevent Activation–

•Guanine nucleotide-dissociation inhibitors (GDIs)

•Inhibit release of GDP

GTPase switch proteins- two large signaling classes

-Heterotrimeric: activated by direct interaction w surface receptors (GEFs)

-Monomeric: activated by GEFs that are activated by surface receptors or other proteins

Four common intracellular second messengers

Cyclic AMP (cAMP)
Cyclic GMP (cGMP)
Diacylglycerol (DAG)
Inositol triphosphate (IP3)

cyclic AMP (cAMP)

-generated from ATP by adenylyl cyclase

-activates PKA

cGMP

-generated by guanylyl cyclase
-activates PKG and specific cation channels

IP3 and DAG

• Both made from PIP2 by phospholipase C
• IP3 - opens channels to release Ca2+ from the ER

DAG

with Ca2+ activates PKC

Calcium ions (Ca2+): intracellullar second messenger

-released from intracellular stores or transported into cell
-activates calmodulin, specific kinases (PKC) and other regulatory proteins

Nitric Oxide (NO): Intracellular/Extracellular second messenger

-formation catalyzed by the enzyme nitric oxide synthase

-highly reactive, only lasts a few seconds

-easily diffuses across membrane

-highly effective for autocrine and paracrine signaling

dissociation constant

measure of receptor affinity for its ligand

synthetic analogs of natural hormones

-may bind much more tightly to the receptor than does natural hormone

-may be more stable

Agonist

-mimics function of natural hormone

-binds to and activates receptor

-induces the normal cellular response to the hormone

Antagonist

-inhibits function of natural hormone

-binds to receptor ligand-binding site but induces no response

-blocks natural hormone binding

-reduces normal physiological activity of hormone

Western blotting

technique that uses antibodies to detect the presence of specific proteins separated by electrophoresis

G protein-coupled receptors (GPCRs)

large family of integral membrane proteins involved in signal transduction (encoded in animal genomes); characterized by their 7 membrane-spanning alpha-helices; utilize heterotrimeric G protein to transmit signals to effector cells

G protein coupled receptors and their 2nd messengers

-bound to membrane by covalently attached lipid chains

-bind to GDP the 3 proteins are tightly associated as a complex

-activation by receptor induces exchange of GDP for GTP

-this alters conformation and releases the Ga and GBy subunits which interact w effector proteins

Mechanism of activation of effector proteins associated with G protein- coupled receptors

•Step 1: Ligand binding induces receptor activation conformational change.

•Step 2: Activated receptor binds to trimeric G protein.

•Step 3: Activated receptor GEF activity stimulates Gα subunit release of GDP.

•Step 4: GTP binding changes Gα conformation
•Dissociates Gβγ (Gβγ subunit activates other effector enzymes in some pathways.)
•Activates Gα

•Step 5: Gα ·GTP activates effector enzyme.

•Step 6: Gα intrinsic GTPase activity hydrolyzes GTP to GDP - dissociates Gα and turns off effector enzyme. (G protein active for minutes or less)

General Signal Transduction Pathway

•
Step 1: Hormone binding to its cell-surface receptor
•
Step 2: Activated receptor (GEF) activates trimeric G protein
•
Step 3: G protein alpha subunit binds to and activates second messenger-generating enzyme.
•
Step 4: Activated enzyme generates multiple second messenger molecules.
•
Step 5: Second messenger activates a protein kinase.
•
Step 6: Kinase phosphorylates and changes activity of one or more target proteins.

Step 6a: Cytosolic target proteins induce changes in cellular function, metabolism, or movement.
•
Step 6b: Target transcription factors induce changes in gene expression.

Signal termination: G protein

-GAP stimulates rapid GaGGTP hydrolysis to GDP
-GaGDP disassociates from effector protein
-effector becomes deactivated

alteration of secondary messengers

-second messengers are either degraded or sequestered

-cAMP phosphodiesterase (PDE)- catalyzes hydrolysis of cyclic bond-- AMP

Deactivation of GPCR

G-protein coupled receptor is phosphorylated and allosterically inhibited by the binding of arrestin

Heart muscle muscarinic acetylcholine receptors activate G protein opening K+ channels:

•Acetylcholine binds to a muscarinic receptor (also activated by muscarine) receptor

•Activated receptor activates a Gαi subunit and its dissociation from the Gβγ subunit.

•Gβγ subunit (rather than Gαi-GTP) binds to and opens a K+ channel (effector protein).

•Increased K+ exit hyperpolarizes the cardiac muscle cell membrane – reduces heart muscle contraction frequency

Termination

Gαi hydrolyzes GTP and rebinds to Gβγ.

adenylyl cyclase

-stimulated and inhibited by different receptor-ligand complexes

Protein Kinase A (PKA):

Two catalytic (C) kinase subunits - transfer terminal phosphate
from ATP to target protein specific Ser/Thr-OH • Two regulatory (R) subunits
• (-) cAMP - bind and inhibit catalytic subunit phosphorylation
activity • (+) cAMP - release active catalytic subunits
• cAMP activates protein kinase A by releasing inhibitory subunits.

glycogen

synthesis and degradation by different pathways-- both regulated by hormon-- induced activation of PKA

increasing cAMP

increased glycogen breakdown, and decreased glycogen synthesis

decreasing cAMP

decreased glycogen breakdown, and increased glycogen synthesis

production of cAMP leads to activation of PKA

•Glycogen synthesis inhibition - PKA phosphorylates-inactivates glycogen synthase (GS)
•Glycogen breakdown activation -

•PKA phosphorylates-activates glycogen phosphorylase kinase (GPK)

•GPK phosphorylates-activates glycogen phosphorylase (kinase cascade)
•GP catalyzes first reaction in glycogen breakdown.

•PKA phosphorylates-activates a phosphoprotein phosphatase (PP) inhibitor - binding to PP prevents de phosphorylation of PKA-phosphorylated enzymes

glycogen synthesis stimulation

PP dephosphorylates-activates GS

glycogen breakdown inhibition

PP dephosphorylates-inactivates GPK

amplification

-single epinephrine molecule binding to one G protein- activates up to hundreds of G proteins (amplification)

-each G protein activates 1 adenylyl cyclase (AC) until G protein hydrolyzes GTP

-AC catalyzes the synthesis of a large number of cAMP molecules while activated (amplification)

-two cAMPs activate two PKA catalytic subunits

non-amplification

two cAMPa activate two PKA catalytic subunits

cAMP/PKA pathway

1. ligand binds to a Gs-protein-coupled receptor, causing a conformational change
2. the alpha(s) subunit releases GDP, binds GTP, moves through the membrane and activates adenylate cyclase
3. activated adenylate cyclase catalyzes the conversion of ATP to cAMP
4. cAMP binds to the regulatory subunit of protein kinase A (PKA), which dissociates from the catalytic subunit, activating it
5. the activated catalytic subunit phosphorylates proteins, causing a response
6.the phosphorylated proteins are rapidly dephosphorylated by serine/threonine phosphatases, terminating the response

Arrestin

enzyme that participates in desensitization of GPCRs by phosphorylating them after they have been activated by ligand binding

Desensitization

blocks active receptors from turning on additional G proteins

-this recruits B-arrestin which binds phosphorylated GPRCs and prevents further activation of trimeric G proteins

Fate of receptors: Arrestin inactivating G protein-coupled receptors

1.Recycled and returned to the cell surface, the cells remain sensitive to the ligand and are said to be resensitized (can respond to new signals).

2.Receptors may be internalized and transferred to lysosomes for degradation: No more receptor, the cell remains desensitized (cannot respond to new signals).

3.May be internalized and serve as intracellular sites for activating novel transduction pathways