BIO 212 Topic 19: Cell Signaling—G Protein Coupled Receptor Signaling

Plasma membrane signal receptor pathways

• second messenger pathway

• phosphorylation cascade pathway

Second messenger pathway

• signaling molecule binds

• binding causes receptor to undergo a conformational change that directly or indirectly activates an effector, or an enzyme that makes a soluble second messenger which carries the signal away from the plasma membrane

• as the second messenger diffuses through the cytosol, it sends a message throughout the cell that elicits a cell response

Phosphorylation cascade pathway

• signaling molecule binds

• receptor conformational change triggers the activation of protein kinases

• protein kinases trigger a series of protein-protein phosphorylations (e.g. a phosphorylation cascade)

• ex: insulin receptor

G protein coupled receptors (GPCRs)

• cell surface receptors that are only found in eukaryotes, pretty much ubiquitously—present in pretty much all types (unicellular, multicellular, plants, protists, animals, whatever) at great abundance

• largest family of signaling receptors; over 800 genes in humans responsible for GPCRs

• conserved 7 transmembrane (7-pass) protein structure among all GPCRs despite variable amino acid sequence

• ligand binding site is in the extracellular loops; cytosolic domains interact with the next proteins in the pathway

• ligand-receptor binding causes conformational changes that increase the GPCR’s affinity for trimeric G proteins

GPCR pathways

• responsible for some metabolic changes, such as mediation of the glucagon pathway, and sensing and responding to the environment

• our sensations of the external world are almost all mediated by GPCRs, including chemoattraction, olfaction, vision, hearing, and taste

Trimeric G proteins

• composed of alpha, beta, and gamma subunits; form heterotrimers

• large number of different combinations of alpha, beta, and gamma subunits; different combinations = different trimeric G protein = different affinities for different receptors and effectors

• about 20 different kinds in mammals

Alpha subunit (trimeric G protein)

• lipid-linked to the plasma membrane (covalently attached lipid domain)

• can move laterally through the plasma membrane until it bumps into the binding site opened by receptor-ligand association

• GDP/GTP binding/exchange mediates activity of the subunit; has intrinsic GTPase activity

• is the subunit that binds the effector enzyme in most (but not all) pathways

Beta subunit

• not lipid-linked to membrane; associated primarily with the gamma subunit

Gamma subunit

• lipid-linked to the plasma membrane

GPCR signal pathway (steps)

• receptor-ligand complex binds GDP αβγ (“off” coformation)

• interaction of the high-affinity G protein with the receptor-ligand complex results in a conformational change in the G protein that results in GDP/GTP exchange on the alpha subunit

• GDP/GTP exchange results in a conformational change that weakens the affinity of the alpha subunit from its partners

• alpha GTP (“on”) dissociates from the βγ subuntis and the receptor-ligand complex

• alpha GTP diffuses through the membrane and binds its partner, the effector (enzyme that generates second messenger)

• alpha GTP binding turns the effector “on,” causing it to produce its second messenger, which then diffuses away from the membrane (is where part of signal amplification occurs—one signal molecule can result in the creation of many second messengers)

• signal pathway shuts itself off

Downregulating GPCR pathway

• alpha GTP hydrolysis

• receptor desensitization (RME); physical removal of the receptor from the plasma membrane

• metabolizing the second messenger

• dephosphorylation of phosphorylated substrates via protein phosphatases

Alpha GTP hydrolysis

• results in dissociation of the alpha subunit from the effector so it can no longer bind substrate, turning off the message

• allows the trimer to reassociate; alpha GDP’s high affinity binding site for beta gamma subunit is open again

• only stops signal for the duration it takes for the trimeric G protein to form and bind the receptor-ligand complex again

GPCR desensitization

• receptor is desensitized by phosphorylation via GRK

• phosphorylated receptor binds arrestin, which arrests/stops the signal and promotes the formation of the clathrin coat by binding the adaptor for clathrin

• PM releases a clathrin-coated vesicle containing the phosphorylated receptor; send to the endosome via receptor-mediated endocytosis (RME)

• endosome sends receptor to a variety of fates

GRK

• GPCR (G-protein coupled receptor) Kinase

• can only act on the receptor-ligand complex, not the receptor alone

Possible fates of the internalized receptor-ligand complex

• sent to ERK (map kinase pathway) or other pathways; signaling crosstalk

• sent to lysosome for degradation

• put in recycling endosome to be sent back to the PM; low pH of the recycling endosome results in dissociation of the receptor from the ligand (ligand will eventually bind again, but for the 15 or so minutes this process takes, the ligand cannot bind the receptor)

Effector examples

• Adenylate cyclase; ATP —> 2nd messenger cAMP

• PLCβ; PIP2 —> IP3 + DAG

Adenylate cyclase

• 12-pass transmembrane protein with the active site facing the cytosol

• when it associates with alpha GTP, it undergoes a conformational change that increases its affinity for its substrate, ATP

• catalyzes conversion of ATP to cAMP (cyclic AMP) and pyrophosphate—pyrophosphate is removed first, producing AMP, and then the alpha phosphate is connected to carbon 3 of the sugar, producing cyclic AMP

cAMP discovery

• plays important role in hormone signaling

• discovered in 1950s when Earl Sutherland observed that in response to certain hormone signals, there was a sudden increase in this molecule (turned out to be cAMP); won Nobel Prize, 1971

• first second messenger to be uncovered

cAMP function

• activates protein kinase A (PKA), which phosphorylates its substrate

• specific substrate and response depends on cell type (the kinds of substrates found in a particular cell) and accessibility of the substrate (sequestering of PKA and subunits by AKAPs)

• responses/targets include (for DIFFERENT cells):

  • plasma membrane (transport)

  • microtubules (assembly/disassembly)

  • triglyceride lipase (fatty acid formation)

  • glycogen synthase (glycogen formation)

  • phosphorylase kinase (glycogen breakdown)

  • nucleus (DNA synthesis, differentiation, RNA synthesis)

  • ER (protein synthesis)

AKAPs

• A kinase anchoring proteins

• targets PKA to a certain substrate

• physically holds the enzyme and substrate nearby or apart

PKA activation

• inactive PKA holoenzyme is composed of four subunits (heterotetramer) with two regulatory subunits and two catalytic subunits

• regulatory subunits prevent binding of substrate to active sites

• catalytic subunits have the active site

• cAMP (four molecules) bind the regulatory subunits, changing their conformations so that they release the catalytic subunits, opening the catalytic subunits’ active sites

cAMP signaling downregulation

• receptor desensitization

• intrinsic GTPase activity of the alpha subunit

• cAMP —> AMP by phosphodiesterases (PDEs) which cut the bond that make it cyclic, via hydrolysis

• dephosphorylation of phosphorylated substrates via protein phosphatases

Phospholipase C.β

• effector enzyme that cleaves phosphatidylinositol-4,5-phosphate (PIP2) into inositol-3-phosphate (IP3, phosphorylated polar head group of PI) and diacylglycerol (DAG; glycerol with two fatty acyl groups in the membrane) via hydrolysis

• activated by interaction with alpha GTP

• noncovalently attached to the cytosolic face of the plasma membrane

Phosphatidylinositol-4,5-bisphosphate (PIP2)

• modified PI

• 2 fatty acyl tails (one saturated, one polyunsaturated) inserted into the membrane

• glycerol

• phosphate head

• inositol, modified to have two phosphate groups—one on C4, one on C5

IP3

• second messenger produced by effector phospholipase C beta (PLC.beta)

• diffuses through the cytosol until it reaches Ca2+ channels inserted into the SER membrane (which are also IP3 receptors)

• receptor is tetrameric and a Ca2+ channel

• IP3 binding results in a conformational change that opens the Ca2+ channel, causing the cytosol to be flooded with Ca2+ (large EC gradient—10^-4 M in the SER vs. 10^-7 M in the cytosol)

Protein Kinase C (PKC)

• is found in the cytosol

• when inactive, it has a C-terminal pseudosubstrate (fake substrate) that is bound by its own active site

• the pseudosubstrate tail is similar enough to the substrate that it binds the active site and prevents other substrates from being phosphorylated, but different enough that it is not phosphorylated itself

• binding of Ca2+ and DAG opens the active site, releasing the pseudosubstrate

• not every PKC isoform binds DAG, but every isoform binds Ca2+ to open its active site so it can phosphorylate substrates

• binding DAG recruits the PKC to the membrane

• substrate depends on cell type/what is available in the cell

PKC functions

• huge variety

• muscle cell differentiation and contraction

• serotonin, dopamine, epinephrine, insulin release

• fat and testosterone synthesis

IP3 signal downregulation

• receptor desensitization

• dephosphorylation of phosphorylated substrates by protein phosphatases

• Ca2+ ATPases in the PM and ER membrane pump Ca2+ back out of the cytosol so that PKC’s active site is inhibited

• Metabolism of IP3 and/or DAG—breaking down or modifying the second messengers terminates or decreases the signal

  • ex: DAG may have a polar head group attached and turn back into a phosphoglyceride

Ca2+ concentration

• cytosolic: ~0.1 microM; low

• extracellular: 1 mM, high

• ER lumen: 0.1 mM, high

• the huge concentration difference between the cytosol and the ECM/ER lumen results in a huge movement/efflux of ions into the cytosol with channels are opened

• low cytosolic concentration is maintained by Ca2+ ATPases (P-type pumps) on the ER and PM

Ca2+ function

• regulates the function of many proteins; influx triggers many events

• some proteins are regulated directly by binding to Ca2+

• some proteins are regulated indirectly by calmodulin, an indirect regulator which modulates the effects of various other proteins via binding—is itself modulated by Ca2+

Regulation of Ca2+ entry into cytosol

• IP3 receptors

• ryanodine receptors

• there are multiple Ca2+ channels and multiple ways the ions can enter the cytosol

• plasma membrane channels are often voltage-gated and open and close based on how polarized the membrane is

Ryanodine receptors

• Ca2+ sensitive Ca2+ channels that are found in electrically-excitable cells, such as neurons and skeletal muscle

Calmodulin (CaM)

• Ca2+ binding protein

• very highly conserved across eukaryotes during evolution, indicating that it plays a highly critical role in the organism

• four Ca2+ binding sites, each of which have a LOW affinity for Ca2+

• low affinity = does NOT bind Ca2+ in low concentrations, but if you drive binding by flooding the cytosol (dramatically increasing concentration), it binds and undergoes conformational change, curling in on itself to form the active form

• active form regulates other proteins