21. G-Protein Coupled Receptors

A family of G proteins that activates cyclic GMP phosphodiesterase in vertebrate rods and cones - the alpha subunit mediates this activation - Belong to family II

Gt (Transducin)

A family of proteins that bind to the R subunit to localize PKA to specific subcellular sites (e.g., nuclear envelope, plasma membrane, cytoskeleton) - Different cells express different PKA anchoring proteins, allowing cell-specific effects of cAMP to occur

A-kinase anchoring proteins (AKAPs)

1.) Binding of ligand to the GPCR causes a conformational change in the receptor that allows the G protein to bind to it. 2.) Binding of the G protein to the activated GPCR alters the conformation of the G protein and the AH domain of the α subunit moves out, allowing GDP to escape. 3.) GTP binding to the α subunit closes the nucleotide binding pocket, which causes conformational changes such that the βγ subunit dissociates from the α subunit 4.) The freed α subunit and the βγ subunit are now able to regulate downstream effector molecules (not shown). 5.) The GPCR stays active as long as ligand is bound so it can activate many G proteins.

Activation of a G Protein By a GPCR

The α subunit is a GTPase. - It contains 2 domains: a Ras domain that is similar to other GTPases and forms one face of the binding pocket and an α helical domain that forms the other side of the guanine nucleotide binding pocket - Other proteins help G proteins with the hydrolysis and exchange of their Guanine nucleotides.

Alpha subunit of G Proteins

Binds to the phosphorylation site on CREB and helps CREB bind the cyclic AMP response elements (CRE)

CREB-binding protein (CBP)

Ca/CaM protein kinases eNOS, nNOS (nitric oxide synthase) PKC Phosphorylase B kinase

Ca2+ and CaM Allosterically Modulate:

An is ubiquitous and abundant protein that can change conformation when it has at least 2 Ca2+(max of 4) bound to it in an almost switch-like manner. - Is a Major Mediator of Ca2+ Signaling - CaM can then bind to other proteins and activate them - CaM is not an enzyme but functions as a subunit of many enzymes and is thus an allosteric activator.

Calmodulin (CaM)

Cholera: ADP-ribosylation of Gs inhibits its GTPase activity, meaning that Gs is permanently in its GTP-bound state and is thus active. - leads to increased cAMP and PKA activity - leads to loss of water ions Pertussin: ADP-ribosylation of Gi prevents it from interacting with its ligand-bound receptors, so Gi remains in the GDP-bound state and is thus inactive. - leads to Gi not inhibiting the basal activity of adenylyl cyclase. - leads to increased cAMP and increased PKA activity.

Cholera and Pertussin toxin and G proteins

A family of G proteins that activates phospholipase C-B - the alpha subunit mediates this activation - Belong to family III

Gq

A family of G proteins that activates adenylyl cyclase and Ca2+ channels - the alpha subunit mediates this activation - Belong to family I

Gs

Structurally, consists of 7 transmembrane helices with an external ligand binding domain (n-term), and an internal G-protein binding domain(c-term). - the Transmembrane domains are 24-25 aa long, hydrophobic α-helical segments - receptor activation by ligand binding triggers the activation of a trimeric G protein that binds GTP or GDP. - There are a ton of these receptors with very diverse ligands. - not a lot of sequence identity between the different types, the similarity lies within the structure and hydrophobic properties of the proteins.

G Protein-Coupled Receptors (GPCRs)

A Family of Signaling Proteins that Bind Guanine Nucleotides and Function as Molecular Switches - the hydrolysis of GTP to GDP is Catalyzed by intrinsic GTPase activity of the G alpha subunit protein - The switching of GDP to GTP is Spontaneous due to high GTP:GDP ratio in cells - Are Trimeric and Attached to the Plasma Membrane (alpha and gamma subunits) by covalently attached isoprenoid (lipid) units. - G Proteins may be physically associated to GPCRs before activation or bind to the receptor after it is activated.

G Proteins

Desensitization of GPCRs Involves Phosphorylation by GRKs (G-Protein Receptor Kinases) and Interaction with Arrestins - GRKs (common one is BARK) phosphorylate the GPCR allowing arrestin to bind - Arrestin interferes with the G-protein-GPCR interaction and prolongs desensitization by preventing dephosphorylation of the receptor. - Receptor sequestration or down-regulation if the signal remains high for very long - b-ARK = b-adrenergic receptor kinase

GPCR Desensitization and BARK

A family of G proteins that inhibits adenylyl cyclase and activates K+ channels - the alpha subunit mediates this inhibition of ad. Cyclase - the beta-gamma complex activates K+ channels - Belong to family II - possible ligands are adenosine and PGE1

Gi

Glucagon is a polypeptide hormone produced in a signaling cell (the α cell in endocrine pancreas) that is secreted in response to low blood glucose levels (stimulus). - An example of a Adenylyl cyclase cascade - PKA activation phosphorylates CREB and glycogen phosphorylase kinase - phosphorylated CREB increases PEPCK txn which increases gluconeogenesis - Phosphorylated glycogen phosphorylase kinase then phosphorylates glycogen phosphorylase which leads to increased glycogen break down - Pathway is turned off at cAMP step by PDE which converts cAMP into 5' AMP. - Pathway is turned off at phosphorylation steps by protein phosphatases.

Glucagon Signalling

Membrane lipid anchors that covalently hold the alpha and gamma sub-unit of G proteins to the cytoplasolic side of the membrane. - Increase the speed and probably the specificity of the G-protein and GPCR interaction. - They are peripheral membrane proteins

Isoprenoid lipid moiety

An uncommon membrane phospholipid found on the cytosolic face of the plasma membrane. -It is the starting point for several second messengers. - cleaved by phospholipase C-B to produce DAG(stays in membrane) and IP3 (water soluble) .

Phosphatidylinositol

A family of enzymes that hydrolyze membrane phospholipids at different positions in the molecule. - Phospholipases of the C subgroup cleave the phosphodiester bond between the glycerol backbone and the phosphate head group. - releasing phosphoinositol from membrane lipids - Gq activates phospholipase C-B

Phospholipases

an Example of a CaM-Activated Protein that stimulates glycogen break down is response to Ca-CaM activation.

Phosphorylase B Kinase

Liver: vasopressin ligand leads to glycogen breakdown Pancreas: Ach leads to amylase secretion Smooth Muscle: Ach causes relaxation in endothelial smooth muscle Blood Platelets: Thrombin causes platelet aggregation.

Physiological Effects Regulated by the Phosphoinositide Pathway

Muscle: there is an increase in Ep causing glycogen break down Liver: there is an increase in glucagon causing glycogen break down Fat: there is an increase in Ep, ACTH, glucagon, and TSH causing triglyceride break down.

Physiological Effects of a Gs-Stimulated Increase in cAMP

A cAMP-dependent protein kinase that activate CREB by phosphorylating it. - PKAs primary targets are metabolic needs. - PKA is a serine/threonine protein kinase. - has a regulatory subunit and a catalytic subunit. - Is Localized within the Cell by Binding to 'Anchoring' Proteins that enhance specificity. - phosphorylates the consensus sequence XR(R/K)X(S/T)B - where X is any amino acid and B is a hydrophobic AA.

Protein Kinase A (PKA)

PKCs are recruited to the plasma membrane by binding to the second messenger DAG (diacylglycerol). - PKCs are activated by Ca2+ and DAG. - PKCs increase growth factor signaling and thus cell proliferation

Protein Kinase C

the GPCR photoreceptor that responds to light by isomerizing a Cis double bond to a trans one in a retinal cofactor. - Cis (inactive) trans (active) forms of retinal. - causes a conformational change that allows the alpha (Gt) subunit to interact with the GPCR.

Rhodopsin

1.) Cytoplasmic Ca2+ is kept low even in the face of high extracellular Ca2+ by ion pumps so that it doesn't form a salt with the cytoplasmic phosphate. 2.) External stimuli can induce a transient increase in cytoplasmic Ca2+ by opening ion channels in the plasma membrane or endoplasmic reticulum. - very short and fast signals.

Roles of Calcium in Signaling

1.) DAG helps activate some PKC family members 2.) DAG is the precursor for arachidonic acid and eicosanoids such as prostaglandins (PGs) - PG-Es regulate many important functions such as pain, vasodilation, suppression of gastric acid release, platelet activation, uterine contraction, inflammation

Roles of DAG in Signaling (2)

1.) The ligand binds to the GPCR; conformational changes in the GPCR activate Gq 2.) The activated Gq activates phospholipase C-β (PLC-β) 3.) PLC-β hydrolyzes phosphoinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3) 4) .DAG helps activate protein kinase C (PKC) 5.) IP3 binds to IP3-gated Ca2+ channels (IP3 receptors) in the endoplasmic reticulum outer membrane, releasing Ca2+ into the cytoplasm. 6.) The Ca2+ helps activate PKC which phosphorylates target proteins, many of which are involved in cell proliferation - Ca2+ and DAG have other signaling roles as well

Steps of the Phosphoinositide Pathway

Adenylyl cyclase enzyme, which is an intrinsic membrane protein is activated by Gs and synthesizes cAMP from ATP. - cAMP is very unstable as it is hydrolyzed by specific phosphodiesterases (PDEs) to form 5'-AMP - Adenylyl cyclase always has some basal activity - Inhibited by Gi

The Adenylyl Cyclase Cascade

Consists of receptors, G-proteins and Effectors - Receptors: Sensory, Adrenergic, Protein hormones, Small peptides and/or Neurotransmitters - G-proteins: 21 α subunit types, 6 β subunit types, 12 γ subunit types - Effectors: Adenylyl cyclase, Phospholipase C-β, Ion channels, and/or cGMP phosphodiesterase (transducin)

The GPCR Membrane Complex

cAMP Is the Allosteric Activator of Protein Kinase A - cAMP binds (at least two molecules) to the regulatory subunit of PKA allowing PKA to release its active catalytic subunits - In the absence of cAMP, the C (catalytic) subunits are bound to R (regulatory) subunits, which inhibits the catalytic activity - No signal amplification

cAMP on Protein Kinase A (PKA)

Is activated by phosphorylation by PKA. - Binds to cyclic AMP response elements (CRE) and increase Txn of the target gene.

cAMP response element binding protein (CREBP)