Chapter 15 - Signal Transduction and G-Protein Coupled Receptors

GDIs (guanine nucleotide dissociation inhibitors)

Inactivate GTPase switch proteins by competitely binding to G-Proteins making it in the permanent off position (make sure this is correct MOA)

Rod Cell Rhodopsin Response (In Darkness)

Step 1. No light absorption means no stimulation of cGMP catalsysis pathway therefore high levels of cGMP in the cytosol. Step 2 high cGMP equals activation of cGMP gated ion channels. Activation of these channels causes influx of Ca+2 and Na+ causing membrane depolarzation to -30mV whhich stimulates release of glutamate which inhibits excitation of neurons and is percieved as darkness.

Second Messenger Targets

targets of second messengers that interact with relay proteins that amplify and direct the signal to the cell interior.

Prostaglandin 1 Hormone

Binds to specific GPCR that leads to adenylyl cyclase inhibition and therefore less cAMP production.

DAG

1,2-diacylglycerol Second Messenger Molecule Produced from PIP2 by phopsholipase C catalysis activates protein kinase C (PKC)

What are the two basic molecular switches used in signal transduction to activate and inactivate proteins in the pathway?

1.Kinase/Phosphatases: Phosphatase (enzyme)- cleave inorganic phosphates Kinases(enzyme)- add phosphate groups via ATP to ADP conversion 2. GTPase Switch Proteins: a. GAP(GTPase Activator Protein) - despite the confusing ass name it actually deactivates G proteins(if their active state is the GTP-bound one) by catalyzing the cleavage of the phosphate in its bound GTP converting it to GDP thereby inactivating it. b. GEF(Guanosine Nucleotide Exchange Factor)- catalyzes removal of GDP and additon of GTP to put protein in its active state(again if active state is GTP bound state)

Ligand

A molecule that binds specifically to another molecule.

GPCR Function and Mechanism in Ion Channels (AcetylCholine)

Acetylcholine binding activates GPCRs allosteric site and GEF activity to eject GDP and attach a GTP activating the Gai subunit. The activated Gai subunit separates the Gby and the Gby subunit goes and activates K+ channels allowing K+ to flow out into ECM. The termination phase not shown invloves intrinsic hydrolysis of the GTP bound to Gai which rebinds the Gby reforming heterotrimeric G PRotein.

B-Arrestin Mechanism for GPCR Termination and Fate

B Arrestin Binds to phosphates attached by GPCRks. Functions as a scaffold for Proteins attached to B arrestin that activate : 1. Endocytotic Pathway(via clathrin and AP2 to be degraded by lysome) 2. Activation of Jun kinase cascade- Activates transcription factors 3. Activation of MAP kinase cascade- Activates transcription factors Arrests GPCR activity after 2 phosphates for rapid signal termination

Glucagon Hormone

Binds a specific GPCR that leads to adenylyl cyclase activation and therefore cAMP production.

ACTH- Adrenocorticotropic Hormone

Binds to specific GPCR that leads to adenylyl cyclase activation and therefore cAMP production.

Epinephrine Hormone

Binds to specific GPCR that leads to adenylyl cyclase activation and therefore cAMP production.

Adenosine(Nucleoside) Hormone

Binds to specific GPCR that leads to adenylyl cyclase inhibition and therefore less cAMP production.

Signal Transduction

Conversion of an impulse pr stimulus from one physical or chemical medium to another

GPCR

G protein coupled receptors. PM TM proteins that bind ligands and have start the signal transduction pathway

Effector Enzymes

Enzymes of the cell membrane that may be regulated by G proteins and that cause biochemical and physiological effects in postsynaptic cells (e.g., by means of second messengers). (Produce Second Messengers)

Hormone Regulation Summary for Glycogen Breakdown or Synthesis

Full pathway based on if the hormone causes activation pathway or inhibition pathway of adenylyl cyclase.

Rhodopsin

GPCR that responds to light and has GEF activity for Gat in Rod cells.

Mechanism of Breakdown and Synthesis of Glycogen

GS- Glycogen Synthase- Synthesizes glycogen by kicking off a UDP that is attached to a glucose with nucleophilic attack of glycogen terminal 4' OH to the glucose 1' carbon attached to the UDP. Activated in response to Hormones that activate adenylyl cyclase GP- Glycogen Phosphorylase- Cleaves 1,4 glycosidic linkages by adding a phosphate to the 1 or 4 carbon position on the glucose sugar. Activated in response to Hormones that inactivate adenylyl cyclase.

Gat

GTP regulated G Protein subunit that binds to y subunits on PDE enzyme which activates PDE

What are the different types of alpha subunits?

Gas- Stimulatory activate effeector enzyme adenylyl cyclase Gai- inhibitory - inhibits adenylyl cyclase Gaq- activates phospholipase C(cleaves acyl chains) Gat- Inhibits phosphodiesterase(leading to enhanced activity of rhodopsin) Ga12/13- Mammal specific activators of cytoskeletal and mitogenic proteins

General Overview of Cell Signaling Steps

Hydrophobic Signal Pathway: 1. Hydrophobic signaling molecule( such as steroids, retinoids, and thyroxine)can freely diffuse through the cell membrane where it meets cytosolic receptor proteins that transport the receptor-signal complex to the nucleus where it can bind to transcription control region in DNA and activate or repress gene expression. Hydrophilic Signal Pathway: 1. Hydrophilic Signaling molecules such as small molecules, peptides, and proteins cannot freely diffuse through the plasma membrane. 2. So these molecules bind to cell surface receptors, and activate the cell signaling pathway through conformational change in the cell surface protein. 3. conformational change induces activation of one or more downstream signal transduction proteins which eventually leads to activation of one or more effector proteins. 4a. These effector proteins can be sent to "effect" proteins involved in cellular metabolism, function, and movement. 4b. alternatively, effector proteins can be sent to the nucleus where they effect gene expression in pathways related to whatever system the signal is used to effect. 5. signal molecule unbinds at end of pathway.

IP3/DAG pathway

IP3 production by PLC spliting of PIP2 results in IP3 Dep gated channels in ER membrane to open cauing high cytosolic Ca+2 which activates PKC ti migrate to PM where it attaches to DAG and is activated allosterically to phosphorylate various proteins in various pathways

How do kinases add phosphates and phosphatases remove phosphates?

Kinases usually add phosphates by dehydrations Phosphatases remove phosphates by hydrolysis

NO Metabolic Pathway

NO produced by NO synthase which is regulated by calcium binding to activate. Ca+2 Regulated by ACh/PLC/IP3 pathway. Production of NO from Arg. and O2 results in NO diffusing into muscle cells where it binds to NO receptor GPCR that ultimately activates guanylyl cylcase that produces cGMP that stimulates PKG. PKG inhibits Ca+2 ER channels which results in low cytoplasmic Ca+2 levels and Ca+2 dependent muscle contraction is inhibited resulting in dilation of walls of blood vessels.

Ca+2

Second Messenger molecule Released/Produced from intracellular stores or transported into cell Activates Calmodulin, PKC(protein kinase C), and other regulatory proteins

RGS9/GB5

Protein complex with GTPase activity that hydrolyzes Gat-GTP complex to GDP thereby inactivating it.

Effector Proteins

Proteins regulated by GPCRs(technically alpha subunit of heterotrimeric G proteins in the direct sense) that produce second messengers

Cytosolic Receptors

Receptors that reside in the cytosol that bind lipid derived hormones. Principally function as transcription factors- once activated they translocate into the nucleus and regulate expression of specific target genes.

Generalized GCPR Signal Transduction Pathway

Step 1: Always starts with ligand binding to GCPR(G protein coupled receptor). Once bound by ligand it activates a heterotrimeric G Protein. (GCPR after binding usually acts as a GEF and switches out its GDP that keeps the G- Protein inactive to an ATP which activates the G-protein.) Step 2: Now activated g protein's alpha subunit binds and activates an effector enzyme. Step 3: Now activated effector enzyme produces secondary messengers (this is defined as amplification of the signal) Step 4: whatever the secondary molecule is diffuses from effector enzyme and activate protein kinases whose main enzymatic function is to phosphorylate other proteins. Step 5: This protein kinase phosphorylates either (a.) proteins associated with the actual pathway(ex: could phosphorylate regulatory proteins that increase activity of enzymes in cellular respiration pathway for example) or (b.) phosphorylate transcription factors that control the rate at which certain genes are expressed(for example in the epinephrine pathway the transcription factor might cause increased translation of new proteins of the cellular respiration pathway to increase ATP production in order to make sure an animal has energy to run away from prey or whatever might have elicited the sympathetic nervous system "stress response"/release of epinephrine hormone. (Ligand-GPCR-Heterotrimeric ATP activated G Protein- Effector Enzyme-Second Messenger Molecules- Regulatory or Transcription Factors)

Heterotrimeric G- Protein Mechanism of action in the GPCR mediated Pathway

Step 1: Binding of hormone to GPCR causes conformaational change that opens the allosteric site of the GPCR allowing for the alpha subunit of the heterotrimeric G Protein anchored to the PM to be able to bind to this site. Step 2: Alpha subunit binds to the GPCR allosteric site Step 3: This binding causes Ejection of the bound GDP that kept it in its inactive state (example of GPCR's GEF activity) Step 4: GTP bound to alpha subunit. This activates the alpha subunit which causes conformational change that ejects the alpha subunit from the GPCR's allosteric site. Step 5: Now active alpha subunit binds to active site of an effector protein thereby activating it. (In most cases Ga - GTP bound to the effector protein activates the effector protein, but textbook states in some pathways that it can inhibit Effector Protein activity.). Recall that effector proteins make second messengers that stimulate regulator proteins and transcription factors. Step 6: Alpha subunit has intrinsic GTPase activity which means as soon as the GTP is bound by the GPCR, it strts hydrolyzing the GTP, therefore it is only bound to the effector protein for a couple of minutes therefore meaning its only activating the effector protein for a few minutes.After hydrolysis of the GTP to GDP the alpha subunit is now rendered inactive thereby inactivating the effector Protein. THe alpha subunit quickly reassociates with the structural beta and gamma subunits of the heterotrimeric G protein resetting the cycle until another hormone binds to the GPCR.

Transcriptional Response in alpha cells in Prancreas in Response to Glucose Levels

Step 1: Catalytic subunits of PKA in response to high cAMP translocate into the nuclear membrane Step 2: Catalytic PKA subunits bind to CREB and after binding it phosphorylates CREB Step 3: Activated CREB binds with co-activator CBP/P300 which attaches to RNA Polymerase during transcription to help CREB anchor to mRNA segment. CREB binds to CRE Regulatory Element that regulates transcription of target genes.

Rod Cell Rhodopsin Response (In Light)

Step 1: Light absorption activates rhodopsin GPCR. Step2: activated rhodopsin binds heterotrimeric g Protein. activated rhodopsin acts as GEF and switches out GDP for GTP. Step 3: this activates Gat subunit which binds to the inhibitory y subunits on the alpha beta Phosphodiesterase (PDE) which allows catalysis of conversion of cGMP to GMP. Step 4: low cGMP concentration keeps cGMP ion gated chaannels closed, therefore membrane is hyperpolarized. Glutamate realease is blocked. (reduction of R is transmitted to the brain as light )

Termination Phase of Rhodopsin signal transduction pathway and the 3 elements that allow it to happen quickly

Termination phase is allowed to happen so quickly because of 1. RGS9/GB5 Complex- has GTPase activity(acts as GAF for Gat subunit) even though Gat subunit has intrinsic GTPase activity. 2. Ca+2 Sensing Guanylate Cyclase Activating Proteins- Sense low Ca+2 levels in cytosol of Rod cell and in response activates guanylyl cyclase to sythesize cGMP to reopen cGMP ion gated channels.(Ca+2 low due to antiporter proteins constantly pumping Ca+2 out of the cell therefore in the light when cGMP gated channels are closed and no Ca+2 influx the Ca+2 concentration is low) Activation of cGMP gated ion channels causes neuron inhibition therefore helping to terminate the cell response quickly to rspond to the ever changing light stimuli the eye is exposed to. 3. Rhodopsin Kinase and Arrestin- a.)Rhodopsin kinase phosphorylates the activated GCPR Rhodopsin proportional to the amount of light Rhodopsin is exposed to. (each opsin has 2 of these serine phosphorylation sites on its cytosol facing C-terminal C4 segment- the more sites phosphorylated the less able R* is to activate Gat. b.) Arrestin- activates when the two or three serine sites on the R* have been phosphorylated. Arrestin binds

Antagonist

a molecule/chemical/drug that inhibits function of natural hormone/drug. (such as how alprenolol inhibits epinephrine function by competitively inhibits GCPR that the epinephrine hormone would bind to.)

Agonist

a molecule/chemical/drug that mimics the action of one of the bodies natural chemicals (such as how isoproterenol mimics epinephrine)

Autocrine Signaling

Type of hydrophilic ligand mediated cell signaling in which the cell excretes its own growth factors that bind to its own cell surface receptors.

Juxtacrine Signaling

Type of paracrine signaling in which requires direct contact (receptor- receptor)

AKAP Action and Mechanism

anchor protein that keeps all of the proteins invloved in cAMP deal in the same place so as to prevent spreading of things to other places.

Second Messengers

small soluble and rapidly diffusing molecukes that regulate activity of specific target proteins usually via allosteric interactions.

CREB Protein

cAMP Response Element Binding Protein(CREB) that binds to promoter of diverse genes ehich in this case is the cAMP Response Element(CRE)

CRE

cAMP response element(CRE) that once binded by response element protein activates transcription of proteins invloved in glucogenesis(Cell Resp.)

Paracrine Signaling

close range cell signaling where one cell releases the ligands that diffuse to the target cell

CBP/P300

coactivator protein that helps locate CREB to RNA poly 2 and to CRE region

cAMP (3'5')

cyclic adenosine monophosphate. Second messenger molecule generated from ATP by action of adenylyl cyclase. Function is it regulates activity of Protein kinase A

cGMP(3'5')

cyclic guanosine monophosphate. Second messenger molecule generated from GTP by action of guanylyl cyclase. Functions to regulate protein kinase G and specific cation channels in rod cells of the eye.

Endocrine Glands

ductless glands of the endocrine system that secrete signaling molecules that go straight into the blood. 7 main Glands: 1. Pineal Gland- Gland in the brain that releases melatonin located beside hypothalamus and thalamus. 2. Pituitary Gland- located below the hypothalamus, and produces oxytocin, vasopressin, anti diuretic hormone, Growth hormone, Thyroid stimulating hormone, adrenocorticotropic hormone, prolactin, and some others. 3. Hypothalamus- produces dopamine, growth hormone releasing hormone, somatostatin, gonadotropin, and others. 4. Thyroid- endocrine gland located where the crycoid cartilage is(adams apple) that produces hormones triodothyronine and thyroxine. 5. Pancreas- located in the middle of the peritoneal cavity behind the stomach that produces glucagon and insulin.(blood glucose regulator) 6.Adrenal Glands- Located right on top of both kidneys and produces symphomimetic epinephrine and nonepinephrine. 7. Ovaries(only in woman) produce estrogen and progesterone 8. testies(only in males ) produce testosterone.

Phosphodiesterase(PDE)

has alpha and beta subunits that catalyze conversion of cGMP to GMP.

Glutamate(in Rod cells)

if produced inhibits neuron excitation (produced in darkness rhodopsin pathway)

Endocrine signaling

signaling in which signaling molecules are synthesized by endocrine glands by the endocrine system. (Long Distance signaling).

IP3

inositol 1,4,5-triphosphate Second Messenger Molecule Produced from PIP2 from phospholipase C Activates Ca+2 channels to release Ca+2 into cytosol

Hormone

ligands that mediate endocrine signaling

Explain the Switching mechanism for Monomeric G Protein (on and off)

monomeric g protein is in active state when bound to GDP. in off state when Intrinsic GTPase activity hydrolyzes gamma phosphate changing bound GTP to GDP separating switch 2 and 1 as shown in figure.

cell surface receptors

receptors that act primarily in the hydrophilic ligand signal pathway that bind hydrophilic ligands which induces signal transduction.

Signal transduction Pathway

series of interactions in which binding by some ligand induces a series of interactions that "transduce" the signal to their appropriate destination.