Chapter 27 Signaling Through TGF-Beta Receptors, Guanylyl Cyclases, and Ion Channels

Question 1

Receptor Serine/Threonine Kinases (TGFβ Receptor Family)

Answer 1

• These are the receptors for a related family of about 33 ligands in humans including TGFbs (transforming growth factor βs), BMPs (bone morphogenetic proteins), anti-Mullerian hormone (AMH, MIS), inhibins, and the activins.
• During development, these proteins regulate pattern formation, influence proliferation, differentiation, tissue remodeling, and cell death
• In adults, they are involved in cell homeostasis, tissue repair and remodeling, and immune regulation

Question 2

Transforming Growth Factor Betas

Answer 2

• Members of the TGFβ Subgroup of the TGFβ Superfamily
• Inhibition of cell proliferation

Question 3

Bone Morphogenetic Proteins (BMPs)

Answer 3

• Members of the BMP subgroup of the TGFβ superfamily
• Osteogenesis
• Chondrogenesis

Question 4

Processing of signaling molecules in TGFβ superfamily

Answer 4

• TGFβ superfamily members are synthesized in a precursor form
• Undergoes dimerization, cleavage, and secretion
• Once secreted, TGFβ superfamily ligands that remain associated with the pro-region and/or other interacting proteins are biologicaly inactive
• Undergoes activation by proteases
• A variety of diverse mechanisms (most poorly understood) result in production of an active form TGFβ superfamily ligand
• Mature TGFβ is 112 amino acids long
• Signaling is typicallly paracrine, can be autocrine, particularly in cance

Question 5

Ligands in TGFβ Superfamily

Answer 5

• Can heterodimerize making interpreting signaling quite difficult
• Ligands are procwessed from a longer precursor to a mature peptide
• May dimerize into homodimersor heterodimers
• Many of the peptides have similar functions and can compensate for each other, making understanding function difficult

Question 6

Signaling Through TGFβ Superfamily Requires 2 Types of Receptors

Answer 6

• Type I Receptors
• Type II Receptros

Question 7

Type I Receptors

Answer 7

Phosphorylate R-Smads

Question 8

Type II Receptors

Answer 8

Bind the ligand, phosphorylate Type I receptors

Question 9

The TGFb/Smad Signaling Pathway

Answer 9

• Receptors for the TGFb superfamily of ligands are composed of two different subunits, i.e. they are heterodimeric. Actually, there are 2 Type I receptors and 2 Type II receptors. Thus, the receptor is a tetramer.
• The type II receptor dimer binds ligand first and then forms a complex with the type II receptor dimer.
• Both receptor subunits possess serine/threonine protein kinase activity.
• After ligand binding, the type II subunit phosphorylates a specific site on the type I subunit to activate its kinase activity
• The type I subunit then phosphorylates latent transcription factors known as Smads.

Question 10

The TGFb/Smad Pathway

Answer 10

Question 11

TGFβ

Answer 11

• Originally described as a tumor-promoting protein (transforming growth factor β).
• However, normally, it is a tumor suppressor and inhibits cell proliferation.
• As tumors progress to metastasis, they become TGFb-resistant.
• Eventually in tumors, TGFb becomes oncogenic, which is why it was called ‘transforming’ growth factor.
• So the consequences of TGFβ signaling are dependent on the cell context

Question 12

Even in the absence of mutations, TGFβ switches from…

Answer 12

tumor suppressor to oncogenic protein as carcinomas progress

Question 13

The Function of TGFβ as a
Tumor Suppressor Protein Can Be Altered in Two Major Ways

Answer 13

• Loss-of-function mutations in the core components of the pathway disable the tumor suppressor effects
  
  • Mutated receptors: Ovarian, esophageal, head & neck, GI, colon, stomach, lung cancers
  • Mutated SMAD4: Pancreas, colon, esophageal cancers
• Downstream alterations that usurp the normal functions of the pathway to cause growth promotion, i.e. this is what happens when TGFβ changes from a tumor suppressor protein to a tumor promoter protein

Question 14

Because TGFβ Affects so Many Functions, It Can Become Tumor Promoting as a Cancer Progresses

Answer 14

Question 15

The Receptor Guanylyl Cyclases: Atrial Natriuretic Peptide (ANP) Receptors

Answer 15

• ANPs are hormones secreted primarily by the heart in response to high blood pressure
• ANPs primarily act by relaxing smooth muscle cells in blood vessels and stimulating excretion of Na+ and H2O. They regulate salt and water balance and thus blood pressure
  
  • Natriuresis is the excretion of sodium in the urine via action of the kidneys, and usually refers to the excess excretion of sodium (diuretic, anti-diuretic)
• The ANP receptors have guanylyl cyclase activity in their cytosolic domains

Question 16

The ANPs

Answer 16

ANPs are synthesized as precursor proteins that are later proteolytically processed to the final active peptide.

Question 17

ANP

Answer 17

atrial natiuretic peptide

Question 18

BNP

Answer 18

brain natiuretic peptide

Question 19

CNP

Answer 19

cardiac natiuretic peptide

Question 20

The Guanylyl Cyclase Receptor Family

Answer 20

Question 21

Receptor Guanylyl Cyclases Make cGMP, Which Activates PKG

Answer 21

Question 22

cGMP Functions as a Second Messenger in a Manner Similar to cAMP

Answer 22

• Most of the actions of cGMP are mediated by binding to PKG (cGMP-dependent protein kinase)
• PKG has both the regulatory and catalytic functions in a single protein
• cGMP is quickly hydrolyzed to 5’ GMP by cyclic nucleotide phosphodiesterases (PDEs), as was cAMP, which helps turn the signaling pathway off

Question 23

The Membrane Guanylyl Cyclase Receptor Signaling Pathway

Answer 23

• One target protein of PKG is the endoplasmic reticulum IP3-gated Ca2+ channel, which is inhibited by phosphorylation.
• Consequently, less Ca2+ is released from the ER and less smooth muscle contraction occurs when PKG is active. Therefore, there is smooth muscle relaxation.

Question 24

Effects of Inactivating Mutations in NPRs

Answer 24

• Mutations in NPRA
  
  • Hypertension and heart failure
• Mutations in NPRB
  
  • Short-limbed dwarfism known as Maroteaux syndrome, which demonstrates its primary role in bone

Question 25

Nitric Oxide (NO)

Answer 25

• NO is a highly reactive and toxic, free radical gas with a half life of ~5 seconds.
  
  • It functions as a signal molecule in biological systems when at low concentrations (pM = 10-12 M)
  • It functions as a killer at higher concentrations (nM = 10-9 M)
• NO is synthesized in cells by a family of enzymes - nitric oxide synthases (NOS) - which use arginine as the N donor and O2 to oxidize N to NO
• Discovery of NO as a biological signal molecule came from studies on how acetylcholine acts to relax smooth muscle cells surrounding blood vessels (vasodilation)

Question 26

Deamination of Arginine to Citrulline by NOS Releases NO

Answer 26

Question 27

A Family of Nitric Oxide Synthases Make NO

Answer 27

• Neuronal constitutive NOS (nNOS)
• Endothelial constitutive NOS (eNOS)
• Induced NOS (iNOS)

Question 28

Neuronal constitutive NOS (nNOS)

Answer 28

• Signaling in the nervous system
• Enzyme activity is regulated.

Question 29

Endothelial constitutive NOS (eNOS)

Answer 29

• Signaling in the vascular system
• Enzyme activity is regulated

Question 30

Induced NOS (iNOS)

Answer 30

• Killing of microbes, viruses (and surrounding cells)
• Gene transcription is regulated (induced).

Question 31

Regulation of NOS Activity

Answer 31

• nNOS and eNOS are activated by the binding of Ca2+-CaM.
  
  • Ca2+ is released by neurotransmitter action, sheer stress, or pressure and binds to CaM (calmodulin). Both nNOs and eNOS release small pulses of NO (pM).
• iNOS transcription is induced in phagocytes (macrophages, monocytes, and neutrophils) primarily by invading microbes or inflammation
  
  • Which elicit the release of interferon-γ or TNFα. As a result, the phagocytes make large amounts of NO (nM).

Question 32

Pathway of NO Signaling in Vasodilation

Answer 32

• Acetylcholine stimulates blood vessel dilation by binding to a GPCR (a muscarinic acetylcholine receptor) on the surface of endothelial cells. This activates Gq.
• Ca2+/calmodulin binds to eNOS to activate it, leading to synthesis of NO.
• NO acts as a paracrine mediator to influence nearby cells
• Binding of NO to intracellular (soluble) guanylyl cyclase activates the guanylyl cyclase, which then makes cGMP. cGMP activates protein kinase G (PKG).

Question 33

Smooth Muscle Relaxation by NO

Answer 33

Question 34

Pathway of NO Signaling in Vasodilation

Answer 34

• NO is small and both hydrophobic and hydrophilic so that it freely diffuses across the membrane of endothelial cells and enters smooth muscle cells
• Inside smooth muscle cells, NO binds to its intracellular ‘receptor’, cytosolic (soluble) guanylyl cyclase, by covalently binding to the Fe of the heme group in the guanylyl cyclase
• This increases activity of the guanylyl cyclase and increases formation of cGMP in smooth muscle
• Increased cGMP works by activating PKG

Question 35

NO Covalently Binds to Fe in…

Answer 35

• Cytosolic (Soluble) Guanylyl Cyclase
• This covalent binding is not readily reversible, so this differs from other signaling systems.

Question 36

Signaling via NO

Answer 36

Question 37

Signaling Through Soluble and Receptor Guanylyl Cyclases

Answer 37

Question 38

Killing by NO: iNOS Is induced in Response to Viruses and Microbes

Answer 38

Question 39

Physiological Effects of NO

Answer 39

Question 40

Pathological Effects of NO

Answer 40

Question 41

Membrane Channels Form Pores Across Membranes

Answer 41

• Channels are membrane proteins that form pores that let the passive movement of ions (ion channels), water (acquaporins), or other solutes to passively across the membrane along their electrochemical gradient.
• Two important properties distinguish ion channels from aqueous pores:
  
  • They show ion selectivity, i.e. they only let a specific ion(s) pass
  • Ion channels are not continuously open. Instead, they are gated (regulated), which allows them to open briefly and then close. In addition, with prolonged chemical stimulation they can go into a closed, desensitized state until the stimulus is removed.

Question 42

Ion Channels

Answer 42

• Ion channels are integral plasma membrane proteins that possess hydrophilic pores highly specific for the transport of inorganic ions. They are selective for specific ions.
• Transport by ion channels is always passive (in the direction of the concentration gradient).
• The permeating ions have to shed most of their water as they pass through a selectivity filter.
• Passage through an ion channel is regulated, i.e. they are not always open.

Question 43

Voltage-gated channels

Answer 43

Respond to a voltage change across the membrane

Question 44

Ligand-gated extracellular ligand channels

Answer 44

typically bind neurotransmitters

Question 45

Ligand-gated intracellular ligand channels

Answer 45

typically bind ions or nucleotides

Question 46

Mechanically-gated channels

Answer 46

are often linked to the cytoskeleton and respond to mechanical stress

Question 47

Signaling Across Synaptic Junctions Is Controlled by Ligand-Gated Channels

Answer 47

• In response to an action potential, secretory vesicles containing neurotransmitter fuse with the membrane and the neurotransmitter is released into the synapse.
• The released neurotransmitter binds to and opens the transmitter-gated channels on the postsynaptic target cell.
• The resultant ion flow alters the membrane potential of the target cell, thereby transmitting a signal from the excited nerve to the target

Question 48

The Acetylcholine Receptors at Neuromuscular Junctions Are Transmitter-Gated Channels

Answer 48

• Acetylcholine binds to 2 structurally and functionally distinct classes of receptors
• One class of receptors are GPCR and activate Gi or Gq. These are often referred to as “muscarinic acetylcholine receptors” because they bind the mushroom toxin muscarine.
• The second class of acetylcholine receptors are ion channels. The channel-forming class of acetylcholine receptors are referred to as “nicotinic acetylcholine receptors” because they bind nicotine.

Question 49

The Nicotinic Acetylcholine Receptor

Answer 49

• Has been studied extensively because it is readily accessible to study because it is at neuromuscular junctions, and there are ~20,000 receptors per μm2 with few other receptors in the same membrane.
• It is composed of five polypeptide subunits that associate to form a channel; each subunit contributes a single transmembrane a helix to form the channel.
• The channel opens when two molecules of acetylcholine bind cooperatively to sites on the extracellular surface of the receptor.
• This causes rearrangement of the a helices to open the pore and allow Na+ to move across the membrane.
• Acetylcholine is rapidly degraded in the synaptic junction and the channel closes within ~1 millisecond.

Question 50

Three Conformations of the Acetylcholine Receptor

Answer 50

• With acetylcholine bound, the receptor still flickers between open and closed states but has a 90% chance of being open.
• This continues until acetylcholine is degraded by acetylcholinesterase and lowers its concentration at the membrane

Question 51

Termination of Synaptic Signaling Requires Rapid Removal of Neurotransmitter

Answer 51

• Most commonly, neurotransmitters are taken up by specific transporters (reuptake transporters) in the presynaptic cell and can be reused.
• Reuptake transporters are often targets of drugs - e.g., the antidepressant Prozac inhibits serotonin uptake (hence, prolonging signaling).
• Many drugs act by binding to transmitter-gated channels: ex. curare blocks acetylcholine nicotinic receptors. Barbituates, tranquilizers (Valium), and sleeping pills such as Ambien bind GABA receptors and help open Cl- channels.