Biochemistry Chapter 8

β-Adrenergic Receptor Kinase (βARK)

- a GRK - recruited to the membrane by the dimeric Gβγ complex Phosphorylation of GPCRs mediated by G protein-coupled receptor kinase provides a docking site on the receptor for a second protein called 𝛃-arrestin, which is a transport protein that binds to the receptor and prevents it from reassociating with the Gαβγ complex GPCR signaling is terminated by recycling the receptors through endocytic vesicles. After ligand binding and dissociation of the GPCR-heterotrimeric G protein complex, G protein-coupled receptor kinase proteins (such as βARK) phosphorylate serine and threonine residues in the GPCR cytoplasmic tail. This process generates binding sites for the endosomal transport protein β-arrestin. After GPCR dephosphorylation in endocytic vesicles, the receptor is either degraded or returned to the plasma membrane for another round of signaling

Classes of Receptor Proteins in Eukaryotes

1. G protein-coupled receptors 2. Receptor tyrosine kinases 3. Tumor necrosis factor receptors 4. Nuclear receptors - All but the nuclear receptors are transmembrane proteins that bind extracellular ligands - All use distinct mechanisms to transduce the downstream signal 5. Ligand-gated ion channels

Consequences of activating a receptor protein

1. Covalent protein modifications: such as phosphorylation and dephosphorylation reactions 2. Protein conformational changes: resulting from high-affinity noncovalent binding interactions between adaptor proteins 3. Altered rates of protein expression, which can occur at the transcriptional level (RNA synthesis and turnover) or translational level (protein synthesis and turnover)

Step-by-Step GPCR Activation

1. Ligand-induced conformational changes in the GPCR 2. Receptor-mediated stimulation of guanine nucleotide exchange (GTP replaces GDP) in the Gα subunit, leading to dissociation into Gα-GTP and Gβγ complexes 3. Regulation of downstream effector processes by the Gα-GTP and Gβγ complexes

Two types of Nuclear Receptor Signaling

1. Steroid receptors - head-to-head homodimers, bind to inverted repeat DNA sequence, 5′-AGAACA-3′ - the ligands are physiologic hormones derived from cholesterol - Ex: estrogen receptor, androgen receptor, progesterone receptor, glucocorticoid receptor, and aldosterone receptor 2. Metabolite receptors - head-to-tail heterodimers, binds to direct repeat DNA sequences, 5′-AGGTCA-3′ - The ligands are derived from dietary nutrients, including vitamins, unsaturated fatty acids, and compounds derived from essential amino acids - Ex: vitamin D receptor, retinoic acid receptors, peroxisome proliferator-activated receptors (PPARs), and the thyroid hormone receptor Nucleotide spacing (# of nucleotides) between inverted repeat sequences for the homodimeric steroid receptors is 3 The heterodimeric complexes formed between RXR (retinoid X receptor) and various subtypes of metabolite receptors bind to direct repeat sequences separated by 1-5 nucleotides, depending on the receptor subtype

Autocrine and Paracrine hormones

Small, secreted peptides that function locally to activate receptor proteins on nearby cells (paracrine) or receptor proteins on the same cell (autocrine).

Protein Kinase A (PKA)

A Downstream Signaling Protein - Active when cAMP levels are high - When cAMP levels in the cell are low, PKA is inactive and exists as an R2C2 tetramer (two regulatory subunits (R) and two catalytic subunits (C))

Insulin Signaling

A cross-linked tetrameric α2β2 complex linked by disulfide bonds. The α subunit is extracellular and contains the insulin binding sites (only one molecule of insulin is required to stimulate receptor signaling); two domains that are abundant in leucine residues; a cysteine-rich region (CR); and a fibronectin domain region (FD) The β subunit anchors the α subunit to the plasma membrane through a transmembrane region (TM) and contains the intracellular tyrosine kinase domain (TK), and a short C-terminal region (CT) Three tyrosine residues in the TK domain (pY1158, pY1162, pY1163) must be autophosphorylated by the ligand-bound receptor before the kinase can phosphorylate other substrates

Nitric Oxide (NO)

A first messenger. A soluble gas (derived from arginine) that activates signaling pathways by diffusing across cell membranes and directly activating signaling proteins. Neuronal input leads to activation of the enzyme nitric oxide synthase, which converts the amino acid arginine into NO and citrulline. This reaction leads to elevated cGMP (Cyclic guanosine monophosphate) levels through activation of guanylate cyclase by NO binding Protein kinase G is activated by cGMP, leading to muscle relaxation and vasodilation Sildenafil prolongs NO-mediated vasodilation by inhibiting the activity of cGMP phosphodiesterase, an enzyme that reduces the level of cGMP by converting it to GMP

Epidermal growth factor receptor (EGFR)

A serum growth factor (signal through paracrine or autocrine mechanisms. Downstream response increases in cell division) Binds to the EGFR and stimulates receptor dimerization on the cell surface EGF is a 53-amino-acid protein that, like insulin, is proteolytically processed from a larger precursor protein to yield an endocrine hormone with three disulfide bridges

Glucocorticoid Signaling

A steroid hormone Required for lung development, carbohydrate metabolism in the liver, inflammatory response, and neuronal signaling in the brain Downstream GR signaling regulates the antiinflammatory pathway in target cells Glucocorticoids cross the plasma membrane and bind to GR (glucocorticoid receptor), releasing it from an inactive chaperonin complex containing the heat shock protein Hsp90 After GR nuclear translocation, the receptor both activates expression of the annexin I gene, which inhibits the inflammatory response, and blocks NFκB-mediated expression of cyclooxygenase-2, a pro-inflammatory gene, by binding to the p65 subunit of NFκB and sequestering it away from the cyclooxygenase-2 gene promoter The net result is reduced inflammation

More than 50% of the pharmaceuticals currently on the market target G protein-coupled receptor pathways. A number of medications meant for treating allergic reactions target the histamine receptor signaling pathway, initiated by histamine. The H1 histamine receptor is coupled to the Gq heterotrimeric G protein. As a new undergraduate researcher in the laboratory, which of the following strategies do you think would be most successful in your efforts to inhibit histamine signaling? More than one option may be correct. Choose one or more: A. addition of H1 histamine receptor antagonist B. activation of phospholipase C β activity C. activation of phosphodiesterase to enhance cAMP levels D. addition of an IP3 receptor antagonist to prevent IP3 binding

A. addition of H1 histamine receptor antagonist D. addition of an IP3 receptor antagonist to prevent IP3 binding Both addition of a receptor antagonist AND addition of an IP3 receptor antagonist will block downstream signaling from Gq-coupled receptors, such as the H1 histamine receptor. Remember, Gq-coupled receptors activate phospholipase C β, triggering cleavage of PIP2 to produce IP3 and diacylglycerol. IP3 binds to its receptor on the endoplasmic reticulum to open calcium channels, triggering a rise in cytosolic calcium.

A low concentration of a first messenger binding its receptor leads to many large changes taking place inside the cell for all signal pathways. The large changes are due to a process called Choose one: A. signal amplification B. signaling specificity C. signaling compartmentalization D. protein kinase amplification

A. signal amplification Signal amplification by second messengers often involves enzymes, which include but are not limited to protein kinases. The overall impact of a small concentration of a first messenger is a growth or amplification of the original signal.

Which of the following best describes a death domain? Choose one: A. the cytoplasmic tail of the TNF receptor, which functions as a protein-protein interaction module B. the active site portion of cysteine-aspartate proteases (caspases) C. the TRADD-FADD complex including procaspase 8 D. the proteolytic target site for CASP8 by CASP3

A. the cytoplasmic tail of the TNF receptor, which functions as a protein-protein interaction module TNF receptors are responsible for pro and anti apoptosis through interactions of the death domain portion of the receptor.

Tasting involves many different cell-signaling processes that ultimately generate nerve signals transduced by membrane depolarization. Sweet tastes result in PIP2 hydrolysis, while salty tastes allow sodium ions to directly alter the membrane potential. What can you deduce about the signaling mechanisms for sweet and salty? Choose one or more: A.Sodium ions directly enter the cells, indicating the signal is transduced by an ion channel. B.Sweet and salty signaling pathways use the same taste receptor molecule, but with different efficacy on the sweet and salty taste center in the brain. C.Sweet utilizes the GPCR signaling pathway, activating phospholipase C. D.Sweet ligands bind to and close sodium channels, which starts a membrane potential.term-43

A.Sodium ions directly enter the cells, indicating the signal is transduced by an ion channel. C.Sweet utilizes the GPCR signaling pathway, activating phospholipase C. The sodium channel involves the epithelial sodium channel receptor to activate the nervous transmission of signal to the brain. This leads to the entry of sodium into the cell, initiating a membrane depolarization. Sweet taste, by contrast, has a set of specific GPCRs that bind to various sweet-tasting compounds (also called tastants). These receptors signal through the classic PLC pathway to activate sodium channels eliciting nervous signaling

G protein-coupled receptors (GPCRs)

Activation results in the dissociation of the heterotrimeric G protein complex This leads to activation of enzymes such as adenylate

Andrenergic receptor agonists and antagonists

Agonists activate receptor signaling by mimicking the natural ligands epinephrine, norepinephrine, or dopamine Antagonists bind to receptors with high affinity and block the binding of physiologic agonists without inducing the structural changes required for signal transduction

Nuclear Receptor Signaling

Also known as intracellular receptors Not bound to membrane Function as transcription factors that regulate gene expression in response to ligand binding Governed by three parameters: 1. cell-specific expression of nuclear receptors and/or coregulatory proteins 2. localized bioavailability of ligands 3. differential accessibility of target gene DNA sequences in chromatin to nuclear receptor binding. Bind to specific DNA sequences in target genes and recruit coregulatory proteins that modulate transcriptional initiation rates The three functional domains of nuclear receptors are 1. the N-terminal domain, which contributes to coregulatory protein binding 2. the DNA binding domain, which binds specific DNA sequences in target genes 3. the C-terminal ligand binding domain, which also encodes coregulatory protein binding sites

Rhodopsin is a well-characterized G protein-coupled receptor that binds retinal. Absorption of light by the retinal molecule allows important neuronal signals to be sent to your brain that enable vision. If you look at a bright light and quickly close your eyes, the retinal molecules quickly turn off that visual signal to the brain. Which of the following do you think is the best explanation for such an observation? Choose one: A. The G proteins, once activated, are separated into α and βγ subunits and this terminates the signal B. Inhibition of the visual signal requires GTPase activating proteins (GAP) binding and GTP hydrolysis C. Guanine nucleotide exchange factors, or GEFs, are slow to respond in order to reactivate the G proteins D. Termination of the visual signal is completely dependent on removal of rhodopsin from the plasma membrane.

B. Inhibition of the visual signal requires GTPase activating proteins (GAP) binding and GTP hydrolysis Inhibition of the visual signal requires GTPase activating proteins (GAP) binding and GTP hydrolysis. The rapid association of these molecules ensures that the signal will be terminated quickly.

One subtype of breast cancer involves the human epidermal growth factor receptor 2 gene (EGF receptor). One in every five breast cancers has a mutation in this gene. Understanding that this is a growth factor receptor gene, which of the answer choices best describes how this type of cancer develops? Choose one: A. Interactions of the mutated receptor, which activates protein kinases, will not impact tumor growth because the ligand cannot bind to the receptor B. Mutations that activate the kinase portion of the receptor result in a receptor that is constantly phosphorylated. This causes constitutive activation of downstream signaling and the resulting cell growth and proliferation C. Mutations that block the receptor from functioning will block cell growth, indicating that this is a loss-of-function gene D. Mutations in the intracellular domain of the receptor will cause Akt to bind without further signaling and regulation of cell apoptotic processes

B. Mutations that activate the kinase portion of the receptor result in a receptor that is constantly phosphorylated. This causes constitutive activation of downstream signaling and the resulting cell growth and proliferation The EGF receptor is activated in a number of ways, which lead to a significant increase in breast cancer cell proliferation. Most of the mutations result in an active receptor leaving the kinase domain active with or without the ligand bound. This leads to constitutive activation of the signaling pathway, resulting in runaway cell growth and cell proliferation---two hallmarks of cancer.

It is important to design an inhibitory strategy that has the smallest probability of inducing side effects. In essence, you want the treatment to be as specific as possible for the allergic reaction. Which of the following strategies has the best chance of avoiding off-target effects (like activation of glycogen synthesis) while preserving the health of the patient? Choose one: A. Include both a Gs inhibitor at the same time as a Gq inhibitor B. Use a specific H1 receptor antagonist to prevent histamine binding C. Add an inhibitor of phospholipase C β only when the patient has low blood sugar so that glucagon synthesis is not stimulated D. Inhibit production of IP3 by phospholipase C β.

B. Use a specific H1 receptor antagonist to prevent histamine binding By binding to the histamine receptor, the antagonist will only inhibit the histamine signaling through this receptor, allowing all other Gq signaling pathways to remain intact The use of antagonists to G protein-coupled receptors, like the H1 histamine receptor, to bind to and block ligand association is a common strategy for preventing or correcting unwanted physiological responses.

Which of the following first messengers is unique because it is not water soluble and does not bind directly to a membrane receptor?Choose one: A. nitric oxide B. estradiol C. acetylcholine D. epinephrine

B. estradiol Estradiol is a water-insoluble hormone/first messenger that is carried by extracellular proteins, crosses the membrane where the steroid binds to a steroid receptor, and will travel to the nucleus to regulate gene activity.

Tumor Necrosis Factor Receptor Signaling

Binding of the small protein tumor necrosis factor-α (TNF-α) to the trimeric TNF receptor, stimulates intracellular pathways with opposing cellular responses (cell death and cell survival), determined by the relative levels of downstream pro-apoptotic and anti-apoptotic signaling proteins Apoptosis: programmed cell death The cell death pathway is mediated by a proteolytic cascade that degrades cellular proteins and kills the cell - The proteins in these pathways are enzymes called cysteine-aspartate proteases (caspases) The cell survival pathway is regulated by a kinase-mediated phosphorylation cascade that activates transcription factors, which induce the expression of genes encoding caspase inhibitory proteins In both pathways, adaptor proteins bind to a region in the cytoplasmic tail of TNF receptors called the death domain (DD), which functions as a protein-protein interaction module

β2-adrenergic receptor (βAR)

Binds epinephrine (ligand) Binding of a ligand happens at the extracellular side of the receptor causing conformational changes on the cytosolic side (intracellular), opening up G protein binding sites that induce signal transduction within the cell These cytosolic conformational changes affect the interactions of the receptor with intracellular signaling proteins, leading to changes in their activity

Hormones

Biologically active compounds that are released into the circulatory system, where they are able to come in contact with hormone receptors contained in target cells. First messengers because they initiate the receptor-activating signal that gives rise to a physiologic response. Hormones can act at a distance through endocrine mechanisms or function locally as paracrine or autocrine signals.

Which of the following correctly describes how phosphorylation of PIP2 to generate PIP3 propagates the insulin receptor signal? Choose one: A. PIP3 is degraded into IP3 and diacylglycerol B. PIP3 is chemically inert, and must be dephosphorylated to PIP2 for downstream signaling to continue C. PIP3 recruits Akt, a PH domain-containing protein, and PDK1 to the plasma membrane D. PIP3 binds to IRS-2, activating PI-3K.

C. PIP3 recruits Akt, a PH domain-containing protein, and PDK1 to the plasma membrane. Membrane-bound PIP3 recruits the Akt pleckstrin homology domain to the plasma membrane, resulting in upregulation of GLUT4 glucose transporters and removal of glucose from the bloodstream

G protein family

ex: Gα Signaling proteins Enzymes called GTPases Cleave GTP to form guanosine diphosphate plus inorganic phosphate (GDP + Pi ) G proteins are in the active conformation when GTP is bound, but when GTP is hydrolyzed, the GDP-bound G proteins are in the inactive conformation

You are culturing mammalian brain cells with standard growth media that includes 10% fetal bovine serum (FBS). FBS has a number of hormones and other components that stimulate cell growth. Over a busy weekend you made a mistake and left the cells in media without FBS. These cells have begun apoptosis while normally cultured cells are growing and surviving just fine. Which might be the reason for the dying cells? Choose one: A. FBS must have included a growth factor signaling to insulin. Loss of this leads to an increase in glucose input into the cells B. Loss of positive growth factors such as TNF-alpha activates the NF kappa B pathway, activating crtical anti-apoptotic genes C. Withdrawal of a positive growth signal induced the cell death pathway of TNF-alpha D. Loss of activators in FBS allows the cells to begin to go into the Go phase, and the cell death is due to an overactive protein kinase A.

C. Withdrawal of a positive growth signal induced the cell death pathway of TNF-alpha Loss of a growth factor over time can induce apoptosis by activating the TRADD death domains, leading to caspase activity. Simple loss of a positive factor will induce many cells to die over a short period of time.

Which binding site most likely represents the area of greatest structural change in response to ligand binding? Choose one: A. cAMP binding B. SOS binding C. heterotrimeric G protein binding D. DNA binding

C. heterotrimeric G protein binding Extracellular ligand binding induces intracellular conformational changes that promote heterotrimeric G protein binding and activation. The G protein is the first intracellular protein associating with the receptor.

The β2-adrenergic receptor is best characterized as which of the following? Choose one: A. none of the above B. mostly β turns C. mostly α helicesterm-41 D. mostly β sheets

C. mostly α helices G protein-coupled receptors traverse the plasma membrane with seven α helices, and are sometimes nicknamed 7-transmembrane (TM) receptors. These helices undergo conformational changes in response to extracellular ligand binding, initiating intracellular signal transduction

Glycogen phosphorylase

Catalyzes a cleavage reaction that releases glucose-1-phosphate (glucose1-P) from the free ends of glycogen polymers. The overall result is the quick release of energy for the organism.

Components of signaling pathway

Consists of protein structural changes initiated by the binding of extracellular messenger molecules (ligands) to receptor proteins, which triggers a cellular response. The name given to the receptor protein reflects the ligand that activates it.

Ligand-gated ion channels

Control the flow of K+, Na+, and Ca2+ ions across cell membranes in response to ligand binding Ex: Nicotinic acetylcholine receptor, which mediates ion transport in response to the neurotransmitter acetylcholine - transmit neuronal signals from nerve cells to muscle cells - Membrane depolarization in nerve axons stimulates release of the neurotransmitter acetylcholine from vesicles that fuse with the plasma membrane near the synaptic cleft Acetylcholine diffuses across the synapse and binds to acetylcholine receptors on muscle cells, triggering membrane depolarization and muscle contraction. - Acetylcholine binding to the α subunits of the nicotinic acetylcholine receptor causes a conformational change in the protein complex This change opens the ion channel and allows Na+ and K+ ions to flow across the membrane and depolarize the cell

Cyclic AMP (cAMP)

Cyclic adenosine monophosphate (second messenger) cAMP signaling pathway: cAMP is produced from ATP by the enzyme adenylate cyclase and hydrolyzed by the enzyme cAMP phosphodiesterase Receptor activation of adenylate cyclase generates cAMP, which in turn binds to and activates a downstream signaling protein called protein kinase A (PKA) The signal is further amplified by PKA through phosphorylation of numerous target proteins, ex: phosphorylase kinase (enzyme 2) Phosphorylase kinase activates a third enzyme in the cAMP signaling pathway called glycogen phosphorylase, the enzyme responsible for the phosphorolysis reaction releasing glucose from glycogen

Both epinephrine (a tyrosine derivative) and glucagon (a peptide hormone) increase glucose export from the liver into the bloodstream. Each ligand binds a different receptor, but both lead to an activation of PKA. How does this happen? Choose one: A. Both receptors bind directly to and activate PKA after the Gsα protein dissociates from the receptor B. The receptors for the two ligands interact before binding to the heterotrimeric G proteins C. Each activated receptor interacts with a unique GTP binding protein that activates PKA D. Both receptors bind and activate the same Gα subunit, Gsα, which indirectly leads to PKA activation.

D. Both receptors bind and activate the same Gα subunit, Gsα, which indirectly leads to PKA activation. The receptor for glucagon and the receptor for epinephrine both interact with the intracellular Gsα subunit of a heterotrimeric G protein. While the ligand-receptor interactions differ between the two, activation of Gsα leads to the same downstream signaling events, and hence the same cellular responses. After the Gsα subunit is activated, it is able to bind with adenylate cyclase, which produces the second messenger, cyclic AMP (cAMP). cAMP levels regulate the activity of protein kinase A (PKA). When cAMP levels are great enough, they bind to the regulatory subunits of PKA, causing them to dissociate from the catalytic subunits of PKA, which can now activate downstream protein molecules by phosphorylating them.

GPCR-Mediated Signaling in Metabolism

Different GPCR-mediated signals can be integrated within a single cell type in response to multiple extracellular stimuli Glucagon receptors and β2-adrenergic receptors activate a shared cAMP-mediated signaling pathway through Gsα, which stimulates adenylate cyclase (AC) activity and the production of cAMP In contrast, epinephrine binding to β2-adrenergic and α1-adrenergic receptors activates parallel pathways using Gsα and Gqα, respectively The Gqα pathway stimulates phospholipase C (PLC) activity, leading to the production of the second messengers DAG and IP3 All three of these signaling pathways converge on target proteins in liver cells that degrade glycogen, leading to increased rates of glucose export

Long-term activation by nuclear receptors differs from the more transient membrane receptor signaling for what reason? Choose one: A. Steroid receptors are antagonistic to most GPCR signaling B. It takes longer for the steroid to diffuse through the cell, while membrane receptor ligands do not require translocation into the cell C. Receptor-mediated signaling takes longer to reverse the phosphorylation caused by kinases, while nuclear protein expression is much faster D. Long-term activation occurs by nuclear receptors activating sets of genes, while membrane receptors involve small second messenger molecules responding quickly, as their signaling occurs by activation of kinases, phosphatases, and other enzymes already expressed in the cell.

D. Long-term activation occurs by nuclear receptors activating sets of genes, while membrane receptors involve small second messenger molecules responding quickly, as their signaling occurs by activation of kinases, phosphatases, and other enzymes already expressed in the cell. It takes longer for proteins to be produced than regulation of existing kinases and phosphatases. However, it should be noted that many of the GPCR and growth factor signaling also terminate in changes in gene expression.

For some diseases like cancer, therapeutic interventions targeting small G protein signaling pathways could be beneficial, especially those interventions that inhibit Ras signaling since, in many cancers, Ras is constitutively active, meaning it is always on. Which of the following might be the best approach to inhibiting growth and division of cancer cells expressing overactive Ras? Choose one: A. increasing GRB2 activity B. inhibiting receptor tyrosine kinases C. increasing MAP kinase activity D. inhibiting Raf activity

D. inhibiting Raf activity Small G proteins like Ras and Raf are important components of tyrosine kinase receptor signal transduction. The ordering of the signals is incredibly important. Since Ras is upstream of Raf, inhibition of Raf will prevent further signal transduction from Ras, such as transcriptional events responsible for growth and division of cancer cells.

Receptor Tyrosine Kinases (RTKs)

Enzymes containing an extracellular domain that binds ligands and an intracellular domain that phosphorylates tyrosine residues in target proteins, including tyrosines within the receptor itself These phosphotyrosine residues create docking sites for intracellular signaling proteins, which function as molecular adaptors

PKA's Role in Epinephrine (α1 Bound)

Epinephrine activation of α1-adrenergic receptor signaling in liver cells stimulates Gqα signaling This activates phospholipase C (PLC), which hydrolyzes PIP2 to generate downstream signaling through the second messengers DAG and IP3 DAG activates the signaling enzyme PKC, and IP3 stimulates Ca2+ release from the endoplasmic reticulum (ER) Activation of phospholipase C signaling by epinephrine leads to a net increase in glucose export as a result of increased glycogen degradation and decreased glycogen synthesis

First messengers

Extracellular ligands that bind to receptor proteins

GPCR Activation Synopsis

GPCRs transmit extracellular signals to the cytoplasm through direct interaction with a membrane-bound protein complex called a heterotrimeric G protein, which consists of one each of Gα, Gβ, and Gγ subunits (Gαβγ) Inactive Gαβγ complex is associated with an unliganded GPCR and contains GDP bound to the Gα subunit Ligand binding to the GPCR induces a conformational change in the GPCR. This change stimulates exchange of GDP for GTP in the Gα subunit The Gα subunit bound to GTP is now activated and dissociates from the heterotrimeric complex After dissociation, both the Gα subunit and the Gβγ complex stimulate multiple downstream signaling pathways The Gα and Gβγ proteins are anchored to the cytosolic side of the plasma membrane by covalently attached lipid moieties

EGF Mechanism

GRB2 binds to pY residues in the EGFR and recruits a GEF signaling protein called son of sevenless (SOS), which binds to and activates a G protein called Ras This binding requires an Src kinase homology-3 (SH3) domain GRB2 contains a SH2 domain that binds to pY residues and two SH3 domains where the SOS binds to it and activates Ras by stimulating the GDP-GTP exchange reaction This combination of SH2 and SH3 domains in GRB2 allows it to function as a bridging protein The activated Ras-GTP protein stimulates downstream signaling pathways. Ras signaling is inactivated by GAPs, such as RasGAP, which stimulates the intrinsic GTPase activity in Ras to generate the inactive-conformation Ras-GDP

Mechanism for terminating GPCR-mediated signals: GPCRs are Controlled by GEFs and GAPs (The G Protein Cycle)

Guanine nucleotide exchange factor (GEF) proteins promote GDP-GTP exchange and activate signaling GTPase activating proteins (GAPs) stimulate the intrinsic GTP hydrolyzing activity of G proteins to inhibit signal transduction - regulator of G protein signaling (RGS): a GAP that functions with G proteins associated with GPCRs

Distinct Downstream Signaling Functions of GPCR

Gβγ: - Stimulate phospholipase A - Regulate ion channels - Regulate receptor kinases Gsα: Stimulatory Gα protein - Activate adenylate cyclase Giα: Inhibit adenylate cyclase Gzα: Regulate neuronal signaling pathways Gqα: Stimulate phospholipases Gtα: Stimulate phosphodiesterases

GTP-coupled receptors bind and interact with intracellular proteins. These proteins, in turn, each have a specific response in cell signaling Description of GPCR signaling:

The action starts with the receptor binding to the ligand and opening the GDP-binding pocket of the alpha subunit. The GDP dissociates and is replaced by a GTP, where the beta and gamma subunits remain as a functional pairing and the alpha subunit separates. Both sets of heterotrimeric G proteins will then interact and regulate their downstream signaling partners.

PIP3 Activation and Insulin Signaling

Insulin receptor signaling activates PI-3K through insulin receptor substrate (IRS) adaptor proteins, leading to the production of PIP3 Once PIP3 is generated by PI-3K, the glycolipid remains in the plasma membrane and serves as a docking site for signaling proteins containing a phosphatidylinositol binding domain called a pleckstrin homology (PH) domain Phosphorylation and activation of Akt by phosphoinositide-dependent kinase initiates a downstream signaling pathway in liver cells that stimulates glucose uptake and glycogen synthesis PI-3K can also be activated by growth factor receptor signaling in some cell types, which regulates a downstream pathway that promotes cell survival

Insulin Signaling Pathway

Insulin receptor tyrosine phosphorylation of IRS (insulin receptor substrate) and Shc (Src homology collagen) proteins, which bind to the receptor cytoplasmic domain through PTB (phosphotyrosine binding) domains, leads to stimulation of two downstream signaling pathways: 1. phosphorylated IRS binds to SH2 domains on PI-3K and activates a downstream signaling pathway that leads to increased glucose uptake and stimulation of glycogen synthesis 2. binding of phosphorylated Shc protein to SH2 domains on GRB2, which activates the MAP kinase pathway, leading to altered gene expression and cell division

Second messengers

Intracellular signaling proteins, which function together to transmit, amplify, and terminate the signal

PKA's Role in Epinephrine (β2 Bound) and Glucagon

Ligand binding to glucagon receptors or β2-adrenergic receptors in liver cells stimulates Gsα signaling, inducing cAMP production by the enzyme adenylate cyclase These upstream signaling events results in the activation of downstream PKA signaling, leading to three distinct metabolic responses: 1. phosphorylation and inhibition of an enzyme required for glycogen synthesis (glycogen synthase) 2. phosphorylation and activation of enzymes involved in glycogen degradation and production of glucose 3. phosphorylation and activation of enzymes involved in glucose synthesis (gluconeogenic pathway) The combined effect of PKA signaling in liver cells is a net glucose export for use as chemical energy by other tissues

Nuclear receptors

Ligand-regulated transcription factors that modulate gene expression through protein-DNA and protein-protein interacting functions

Cell signaling pathway

Linked set of reactions that are initiated by ligand-induced activation of a receptor protein and terminated by a cellular response. - Early steps in a signaling pathway are called upstream events - Later steps, are referred to as downstream events The creation of second messengers by an upstream signaling protein results in signal amplification through the activation of one or more downstream target proteins

Ras

Member of G protein signaling molecules (Gα) of heterotrimeric G proteins Anchored to the cytoplasmic side of plasma membranes by a covalently attached lipid moiety Activated by GEF proteins Contains an intrinsic GTPase activity that controls its signaling function and is regulated by GAPs

G Protein-Coupled Receptor Signaling

Most of the G protein-coupled receptors (GPCR) are involved in sensory perceptions such as: • Vision • Taste • Smell - Contain seven transmembrane α helices and are oriented with the N terminus on the outside of the cell and the C terminus on the inside. The N-terminal domain of most G protein-coupled receptors contains one or more carbohydrate functional group (they contribute to their molecular recognition properties) - Called "serpentine receptors" because the seven transmembrane helices traverse in and out of the plasma membrane

Sensory Perception Mechanisms and GPCR

Neuronal transmission by visual, olfactory, and gustatory cells requires GPCR-mediated signaling through specific Gα proteins These proteins regulate intracellular levels of second messenger signaling molecules Light absorption by the retinal group in rhodopsin activates Gtα, which stimulates a phosphodiesterase and cGMP hydrolysis, causing ion channel closure and neuronal signaling Odorant binding to olfactory receptors activates Gsα, which stimulates adenylate cyclase and cAMP generation, causing ion channel opening and neuronal signaling Binding of sweeteners to taste receptors on tongue cells activates Gqα, which stimulates phospholipase C and the generation of DAG and IP3, causing ion channel closure and neuronal signaling

Cancer and Ras Mutations

Oncogenes: cancer-causing genes Somatic mutations: Human cancers can be the result of dominant gain-of-function mutations in cell signaling genes or recessive loss-of-function mutations in the same cell. Cancer caused by a dominant oncogene mutation requires one copy of the two genes be defective in a cell Cancer caused by a recessive oncogene mutation requires that both copies of the gene be defective in the same cell Cells with a recessive mutation in only one gene copy often have a normal phenotype. The most common oncogenic Ras mutations lead to defects in the intrinsic GTPase activation and thereby block Ras protein inactivation Dominant Ras mutations in the GTPase domain lead to chronic stimulation of the MAP kinase signaling pathway, even in the absence of growth factors

Cyclic GMP (cGMP)

Or: 3′,5′-cyclic guanosine monophosphate A second messenger Produced from guanosine triphosphate (GTP) by the enzyme guanylate cyclase

Endocrine hormones

Produced by glands that secrete hormones into the circulatory system. Come in contact with receptor proteins located in the cells of target tissues.

Ras Protein Signaling

Ras transmits the EGFR signal downstream to specific target proteins through the stable activation of a phosphorylation cascade mediated by a mitogen-activated protein kinase (MAP kinase) pathway The first kinase in the pathway is Raf: phosphorylates target proteins on serine and threonine residues Once activated by phosphorylation, Raf phosphorylates serine residues on MAP/ERK kinase (MEK), leading to MEK phosphorylation of extracellular signal-regulated kinase (ERK). Phosphorylated ERK forms a homodimer that translocates to the nucleus, where it phosphorylates several target proteins These proteins function as transcription factors and regulate gene expression Signaling through the EGFR pathway is terminated by RasGAP inactivation of Ras function and by phosphatase enzymes that remove the activating phosphates from MAP kinase proteins and transcription factors

Mechanism for terminating GPCR-mediated signals: Receptor desensitization after dissociation of the Gαβγ complex

Regulatory proteins, called G protein-coupled receptor kinases (GRKs), phosphorylate the GPCR cytoplasmic domain on serine and threonine residues, which marks it for recycling

SH1, SH2, SH3

SH1: A tyrosine kinase domain SH2: A domain forming a deep pocket with a positively charged amino acid to accept phosphotyrosine SH3: A large domain of approximately 70 amino acids that bind proline-rich sequences of adaptor proteins

Diacylglycerol (DAG), Inositol-1,4,5-trisphosphate (IP3), & Calcium ion (Ca2+)

Second messengers in signal transduction pathways Phospholipase C hydrolyzes the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to form both DAG and IP3 The enzymatic activity of phospholipase C is regulated by activation of a membrane receptor Once PIP2 is hydrolyzed by phospholipase C, the newly generated second messenger DAG binds to and activates protein kinase C (PKC), which phosphorylates downstream targets The second messenger IP3 activates calcium channels located on the endoplasmic reticulum, leading to the release of Ca2+ from the endoplasmic reticulum and a rapid increase in cytoplasmic Ca2+ levels Intracellular signaling by cytosolic Ca2+ involves activation of a Ca2+ binding protein, calmodulin (a signaling protein that binds to and activates a wide variety of target proteins) PKC proteins bind two or more Ca2+ ions (in the C-2 domain of PKC), calmodulin binds four Ca2+ ions and undergoes a large conformational change when it binds to target proteins The activities of PKC and calmodulin are both regulated by Ca2+ binding

Second messenger

Small, nonprotein intracellular molecules whose functional role is to amplify receptor-generated signals.

Two-step model of EGF function

Step 1: - binding of an EGF molecule to each of two EGFRs induces receptor dimer formation - Dimerization of the EGFRs leads to activation of kinase activity in (EGFR1) and subsequent phosphorylation of tyrosine residues in the cytoplasmic tail of (EGFR2) Step 2: - phosphorylation of the five tyrosine residues in EGFR2 induces a conformational change (in the cytoplasmic domain) in the receptor dimer, resulting in activation of the EGFR2 kinase domain and tyrosine phosphorylation of EGFR1 residues

Activation of TNF Receptor Complexes

TNF is a homotrimer Activation of trimeric TNF receptor complexes by TNF-α binding induces a conformational change that promotes exchange of an inhibitory protein, silence of death domain, (SODD) for a downstream signaling protein, TNF receptor-associated death domain, (TRADD) The binding of TRADD proteins to TNF receptors creates an activated adaptor complex that binds additional downstream signaling proteins

Apoptosis

TNF signaling pathway splits (branches) at the level of TRADD binding to the receptor In the apoptosis branch of the pathway, the DD region of TRADD forms a complex with the DD region of the adaptor protein Fasassociated death domain (FADD) The TRADD-FADD complex then recruits procaspase 8 to the TNF receptor complex using a second protein-protein interaction module called the death effector domain (DED) It then stimulates an intermolecular reaction in procaspase 8 that generates the proteolytically active caspase 8 (CASP8) enzyme Caspase 8 cleaves procaspase 3 to generate caspase 3 (CASP3), the "executioner" caspase, which then degrades key regulatory molecules to kill the cell quickly and efficiently

Production of PIP3 in the cell is a downstream consequence of insulin receptor signaling, and breakdown of PIP3 can shut the system off

The production of PIP3 from PIP2 is facilitated by the action of PI-3K, which transfers a phosphate from ATP to PIP2. The reverse reaction is catalyzed by PTEN, a phosphatase, which through the input of water, releases inorganic phosphate as a by-product of the reaction

The G Protein Cycle

The sequential stimulation of G protein signaling by GEF activity, with subsequent activation of its intrinsic GTPase activity by GAPs 1. Ligand stimulation of the GEF function of a GPCR 2. Dissociation of Gα and Gβγ from a GPCR 3. GAPs such as RGS2 stimulate the intrinsic GTPase activity of Gα 4. Reassociation of Gαβγ with a GPCR

Tumor necrosis factor (TNF) receptors

Transmit extracellular signals by forming receptor trimers, which direct the association of cytosolic adaptor protein complexes Regulates signaling pathways that control inflammation and apoptosis (programmed cell death) Contain a protein binding module called a death domain

Receptor Tyrosine Kinase (RTK) Signaling

Transmit extracellular signals by ligand activation of an intrinsic tyrosine kinase function found in the cytoplasmic tail of the receptor Activated RTKs are dimers, so the kinase phosphorylates tyrosine residues on the paired cytoplasmic domain through an intermolecular reaction Phosphorylates downstream signaling proteins that bind to the RTK phosphotyrosines Some of these targets for RTK phosphorylation serve as adaptor proteins, which function as molecular bridges to bring together other proteins Many of these RTK target proteins are also kinases, so their phosphorylation and activation establishes a relay signal between the receptor and a downstream phosphorylation cascade Two RTK signaling pathways: 1. Epidermal growth factor receptor (EGFR) 2. Insulin receptor

Signal transduction

Transmits extracellular signals across the plasma membrane and throughout the cell. Changes information into a chemical signal. Often ends with covalent or noncovalent modification of intracellular target proteins. Ex: Flux through metabolic pathways Ion flow through the plasma membrane Cell mobility Gene expression

Serotonin is an important hormone that is involved in mood and appetite. It acts on receptors nearby the target cell. Serotonin is an example of a(n) __________hormone a. autocrine b. endocrine c. paracrine d. juxtacrine e. novacrine

b. endocrine

Which of the following molecules is a first messenger? a. Ca2+ b. insulin c. cGMP d. IP3 e. GRB

b. insulin

Please put the steps of a signaling pathway (from the list below) in order: 1) Intracellular signal sent to target protein 2) Termination of the signal 3) Ligand binds to the receptor 4) Binding causes a conformational change. a. 1,2,3,4 b. 3,2,4,1 c. 4,3,1,2 d. 3,4,1,2 e. 2,1,4,3

d. 3,4,1,2

For G protein-coupled receptors, which of the following is a plausible termination method? a. Gα and Gβγ subunits associate b. β-arrestin binds to the GPCR complex c. The ligand dissociates from the receptor d. GAP proteins do not bind to the Gα subunit e. all of the above

e. all of the above

Nicotine, a chemical found in cigarettes, is a receptor agonist of acetylcholine. It works by binding to the receptor and controlling the flow of Na+ and K+ ions. This is an example of a _________ a. G protein-coupled receptor b. receptor tyrosine kinase c. tumor necrosis factor receptor d. nuclear receptor e. ligand-gated ion channel

e. ligand-gated ion channel