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There are up to ___ cells in the body and ___ cell types with specific functions. Cells need to be able to communicate and this occurs through ___.

100 trillion; 1,000; cell signaling


What two things does cell signaling require?

1. A signaling molecule (hormone, NT, etc)
2. Receptor protein


What are the two types of receptor proteins? Describe them.

1. Cell surface receptor protein - binds hydrophilic signals that cannot pass through the membrane
2. Intracellular receptors - binds hydrophobic signals that can pass through the membrane


Different types of signaling are defined by the ___ of the signaling molecule.



What ware the 5 types of signaling as defined by the source of the signaling molecule?

1. Contact-dependent: signaling cell contacts a target cell's receptor via a membrane bound signal.
2. Paracrine - secreted signal affects local cells
3. Autocrine - secreted signal affects local cells of the same type or itself
4. Synaptic - neurotransmitters communicate across a synapse; this is very fast.
5. Endocrine - affects cells far away, travels in the blood; this is very slow


A cell will respond to a signaling molecule only if it has a specific ___ for that molecule.



Receptors must have a high affinity for their signals - why?

The concentration of hormones in the blood is relatively low.


Cells depend on multiple extracellular signals - true or false.



A hormone can have different effects in different types of cells depending on what two things?

The type of receptor present and how the cell is programmed to respond


What is an example of the different effects a hormone can have in different types of cells?

Acetylcholine can:
1. Bind to nicotinic receptors on skeletal muscle cells to cause muscle contraction.
2. Bind to M2 receptors of heart muscle cells to decrease the rate and force of contraction.
3. Bind to M3 receptors of salivary gland cells to cause secretion.


Some hydrophobic signals pass through the membrane (passively or via transport) and activate ___. Give an example.

Nuclear receptors; steroid such as cortisol, estradiol, thryoxine, testosterone, vitamine D3, retinoic acid


When steroid hormones bind to their receptors, the receptors typically ___ and then bind to specific ___ to regulate transcription.

Dimerize; DNA sequences


The proteins first created by the binding of hormones are known as ___. They are sometimes ___ that can stimulate gene transcription.

Primary-response proteins; transcription factors


Sometimes the transcription factors created primarily can stimulate transcription of other genes (this is known as a ___ response) or turn off the primary response (this is known as ___).

Delayed secondary; feedback inhibition


The members of the nuclear receptor super family are structurally related and contain what three domains?

1. DNA-binding domains
2. Hormone-binding domains (C-terminal domain)
3. Transcription regulating domains (N-terminal domain)


Inactive nuclear receptors have bound ___ proteins near the ligand (hormone) binding domain. When a hormone binds, ___ changes result in dimerization, the release of these proteins, and the binding of coactivators or corepressors that regulate ___ of specific genes.

Inhibitory; conformation changes; transcription


What are GPCRs?

G-protein-coupled receptors; proteins with 7 transmembrane helices to which signaling molecules bind at the extracellular N-terminus. The cytoplasmic side of the protein interacts with G-proteins.


The G-protein itself is a ___ made up of what subunits? Which subunits are tethered to the membrane? Which subunit binds GDP in the inactive state?

Hetertrimer; alpha, beta, gamma; alpha and gamma; alpha


Describe the general process of activation and inactivation of G-proteins.

1. Signal molecule binds to GPCR.
2. GPCR activates G-protein by stimulating release of GDP, followed by binding of GTP.
3. Activated G-protein (w/GTP) dissociates from the receptor and the alpha subunit dissociates from the beta-gamma subunits. The alpha(GTP)-subunit and/or the beta/gamma-dimmer can activate specific enzymes or ion channels.
4. Hydrolysis of GTP to GDP by alpha-subunit GTPase activity occurs, releasing a phosphate ion. The alpha-subunit and target protein dissociate.
5. The inactive alpha subunit reassembles with the beta-gamma complex to reform an inactive G-protein. This can be reactivated.


What can accelerate inactivation of G-proteins?

Target enzymes or ions channels, or by RGS (regulators of G-protein signaling)


There are many different G-proteins made of various alpha, beta, and gamma subunits. These G-proteins control the activities of various enzymes and ion channels. What are the three main G-proteins and what do they primarily do?

1. Gs - alpha-s mediates activation of adenylyl cyclase
2. Gi - alpha-i mediates inhibition of adenylyl cyclase, beta-gamma activates K+ channels
3. Gq - alpha-q mediates activation of phospholipase C-beta


What happens when a signaling molecule binds to a GPCR that is coupled to Gs?

The G-alpha-s(GTP) subunit activates adenylyl cyclase. This enzyme produces cyclic AMP (cAMP) by removing 2 phosphates from ATP. cAMP is hydrolyzed and inactivated to 5'-AMP by cAMP phosphodiesterase. cAMP affects the cell by activating protein kinase A (PKA).


How does cAMP activate PKA?

cAMP binds to sites on 2 regulatory subunits of PKA and releases 2 activated catalytic subunits which can phosphorylate other proteins.


Where does PKA phosphorylate proteins?

Puts a P from ATP on specific Ser or Thr aa in select proteins, altering their activity.


What is the amino acid sequence that allows PKA to phosphorylate a protein?

Arg-Arg-X-Ser(or Thr)-Y(hydrophobic)


Many hormones stimulate cAMP production in their respective target tissues. The consequences of increased cAMP are different in different ___. Why?

Cell types; this is due in part to the fact that their are different proteins present in each cell type available for phosphorylation.


How are phosphorylated proteins dephosphorylated?



How does PKA regulate gene transcription?

Phosphorylation of a TF called CREB in the nucleus


Describe the process of transcription regulation with CREB beginning with activation of a GPCR.

1. Signal molecule activates GPCR
2. Gs alpha-subunit activated
3. Adenylyl cylcase activated
4. cAMP created
5. cAMP activates PKA
6. PKA flows into nucleus via pores
7. PKA activates CREB
8. CREB binds to CRE (cAMP response element) sequence
9. CBP (CREB-binding protein) associates with CREB
10. Transcription occurs


How can GPCRs become desensitized?

Phosphorylation on their cytosolic loops and tails by kinases such as GRK. The phosphorylated receptor is recognized by an arrestin protein that binds and prevents the receptor from interacting with G-protein. Arrestin can also direct the receptor to clathrin-coated pits where it can be endocytosed. The receptor can be transiently sequestered, dephosphorylated, and sent back to the plasma membrane, or it can be ubiquitinated and degraded.


Bound arrestin can also activate additional ___.

Signal transduction pathways


Describe the example of cholera toxin.

Cholera toxin contains an enzyme subunit that uses NAD+ to put an ADP-ribose group on Arg-201 of G-alpha-s (ADP-ribosylation). This inactivates GTPase activity of G-alpha-s so that it cannot cleave bound GTP. Continuous activity activates adenylyl cyclase, produces lots of cAMP in intestinal epithelial cells, which activates PKA, which phosphorylates CFTR. Cl- is secreted, Na+ aborption is inhibited, and H2O follows, leading to diarrhea.


Some signaling molecules bind to GPCR and activate Gi. The mechanism is analogous to activation of Gs, but the G-alpha-i-GTP subunit ___ adenylyl cyclase, ___ cAMP. What can the G-beta-gamma-i dimer do?

Inhibits; decreasing; activate certain K+ channels


The activated alpha-subunit of the G-q-protein activates membrane associated enzyme ___. This cleaves the minor membrane lipid ___ to form what two products?

Phospholipase C-beta; phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2); diacylglycerol; IP3 (inositol 1,4,5-trisphosphate)


How is PI(4,5)P2 synthesized?

PI is phosphorylated by a PI kinase to PI(4)P. PIP kinase phosphorylates this to PI(4,5)P2.


What does diacylglycerol do?

Activates a Ser/Thr protein kinase, protein kinase C.


What does IP3 do?

Binds to specific calcium channels in the ER, allowing calcium to move along its concentration gradient from ER lumen into the cytosol, increasing cytosolic calcium.


Describe the process of activation of Gq.

1. Signal binds to and activates GPCR.
2. GPCR activates Gq
3. Gq activates phospholipase C-beta
4. Phospholipase C-beta cleaves PI(4,5)P2 to IP3 and diacylglycerol
5. IP3 opens calcium-release channels in the ER, diacylglycerol activates PKC (sometimes calcium can further activate PKC)


Calcium is a key signal that controls many cellular functions. Concentration of calcium in the cytosol is kept very ___ by mechanisms that move calcium ___ of the cell or into the ___/___.

low; out; ER/mito


What are the 5 ways calcium concentration is kept low in the cytosol?

1. Sodium-driven calcium exchanger (3 sodium enter the cell down its gradient, 1 calcium exits against its gradient
2. Calcium ATP-ase pump (uses ATP to pump calcium out of the cell)
3. Calcium-pump in ER membranes (uses ATP to pull calcium out of the cell into the ER)
4. Calcium binding molecules in cytoplasm
5. Calcium import into the mitochondria via H+ symport


The careful regulation of calcium allows for rapid changes in cytosolic calcium concentration by opening calcium channels in the plasma membrane or ER. Opening of calcium channels on the ER is often followed by what?

Opening of calcium channels in the plasma membrane (sustains elevated cytosolic calcium levels or replenish ER stores)


Changes in cytosolic calcium concentration vary over ___ and ___.

Time; space


Small, localized increases in cytosolic calcium concentration can lead to... What causes this?

...waves of calcium release that move across the cell. This is caused by calcium-induced calcium release, where IP3-induced increase of calcium at one point can stimulate opening of nearby channels. Eventually, channels close by feedback inhibition, terminating the wave.


Cells can respond to a signal that activates Gq by producing a series of waves or spikes resulting in ___. What signal can do this? Note that the frequency increases, not the ___.

Calcium oscillations; vasopressin; amplification


Many effects of calcium are mediated by ___, a small protein with ___ calcium ion-binding sites.

Calmodulin; 4


When at least ___ sites are filled, the calmodulin complex can bind to enzymes and membrane transport proteins and alter their activity. What is one such protein?

Calcium-calmodulin-dependent protein kinases (CaM-kinases)


What is CaM-kinase II?

Complex of 12 subunits that are partially activated by binding of calcium-calmodulin. It can then autophosphorylate (phosphorylate itself) to become fully active.


What happens to CaM-kinase II when calcium decreases?

Calmodulin dissociates, but the enzyme retains significant activity until a phosphatase removes the phosphate. This creates a memory trace.


Autophosphorylation of CaM-kinase II also increases binding affinity for ___, which delays its release. What does this do?

Calcium-calmodulin; allows even a small calcium signal to quickly and fully reactivate the enzyme.


CaM-II kinase has two domains. What are they and where are they found?

1. Catalytic domain (N-terminal domain)
2. Inhibitory domain (C-terminal domain)


CaM-kinase II can interpret the frequency of calcium oscillations. If there is enough time between spikes, the phosphatase will have a chance to remove the phosphate and ___ the enzyme. If the frequency is faster, all of the enzyme will not be completely inactivated. What happens?

Inactivate; More of the enzyme will become activated over time


The faster the oscillations, the higher the...

...activity of CaM-kinase activity


Fully activated enzymes can be maintained as such with low frequency calcium signals. Why?

High affinity of the autophosphorylated enzyme for calcium-calmodulin


The effect a signaling molecule has on a cell depends in part on which set of proteins are...

...available for phosphorylation.


The hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) have ___ different GPCRs. These multiple GPCRs couple to different G-proteins and have different tissue locations and functions. What are the 3 types, the respective GPCR subtypes, and the principal G-proteins?

1. Alpha1; alpha1A, alpha1B, alpha1D; Gq for all
2. Alpha2; alpha2A, alpha2B, alpha2C; Gi, Go for all
3. Beta; beta1; Gs, beta2; Gs, beta3; Gs, Gi, Go


The different adrenergic receptor subtypes have hormone binding sites that are structurally ___ from one another. What is the consequence of this?

Dissimilar; drugs can be designed to bind specifically to certain receptors and not others


What are the two major types of drugs that affect hormone binding sites?

1. Agonist - drug activates receptor
2. Antagonist - blocks ability of hormone to bind


What is nitric oxide (NO)?

Gas used as a signaling molecule


How is NO produced?

By nitric oxide synthase (NOS) enyzmes from arginine, oxygen, and NADPH.


Endothelial NOS (eNOS or NOS3) is activated by what?

Calcium-calmodulin in response to hormones that activate Gq


Describe the effects of NO.

1. NO passes through membrane, binds to/activates soluble guanylyl cyclase.
2. Soluble guanylyl cyclase produces cGMP
3. cGMP activates PKG
4. PKG phosphorylates other proteins


NO is involved in signaling from endothelial cells, which line the lumen of ___ to the underlying ___ cells, which control vessel ___.

Blood vessels; smooth muscle; diameter


NO causes smooth muscle relaxation, which does what?

Increases dilation, increases blood flow, decreases blood pressure


NO formation can be stimulated by hormones such as ___ or ___.

Bradykinin; acetylcholine


Describe what happens after bradykinin binds?

1. GPCR activates Gq
2. Gq activates phospholipase C-beta
3. Phospholipase C-beta produces IP3
4. IP3 increases calcium concentration in cytosol
5. Calcium binds calmodulin
6. Calmodulin helps form NO from NOS and Arg (all occurs in the endothelium)
7. NO travels to smooth muscle, binds to and increases soluble guanylyl cyclase
8. Guanylyl cyclase creates cGMP
9. cGMP activates PKG
10. Muscles relax


___ bypass the endothelium and cause production of NO in smooth muscle.

Nitrovasodilators (sodium nitroprusside, nitroglycerin, isosorbide dinitrate)