8/14/01

Lecture #18 - Signal Transduction

Signal transduction is the process of converting an extracellular signal into a cellular response. We'll concentrate on signals that modify transcription.

Examples of responses within a cell

• gene expression --> differentiation (undifferentiated cell --> specific fate)
• cell division
• growth of cell- changes in metabolism
• changes in cell shape- usually by changing the cytoskeleton (microtubules, actin filaments, or intermediate filaments)
• cell shape changes are important for cell movement- for example, cell migration in development (in the embryo, certain classes of cells migrate in different directions- change shape, for filiopodia or lamellipodia and move along substrate)
• programmed cell death
• ** All of these different activities are affected by gene expression. It's one way of controlling all of the behaviors.

Some signals can enter the cell

1. small ions- K, Na, and Cl enter the cell to exert an effect
2. small molecules like glucose (through transporters or channels)
3. endocytosis- the cell envelopes large proteins to bring them inside
4. steroid hormones- diffuse through the membrane because made of lipid-soluble cholesterol derivatives
   • steroid hormone receptors are in the cytosol and are transcription factors. They are not proteins on the surface of the cell in the plasma membrane.
   • the hormone/receptor complex can bind DNA and elicit a change in transcription
   • it binds to a response element in the cis-regulatory region of the gene being expressed

** Most extracellular signals have a major effect on cell behavior without entering the cell.

Nomenclature

ligand - extracellular signal that interacts with a receptor on cell surface

some examples are:

• peptide hormones- small peptides, not lipid-soluble. examples are insulin, growth hormone, and FSH.
• growth factors- small proteins that stimulate the cell to divide. examples are EGF (epidermal growth factor), PDGF (platelet-derived growth factor), and interleukin-2.
• neurotransmitters- molecules released from one nerve cell that have a direct effect on a nerve cell or other cell type. examples are epinephrine (adrenaline), histamine, and dopamine.

The ligand does not enter the cell, but interacts with a receptor (a protein that spans the plasma membrane one or more times). One or more domains of the receptor is on the extracellular space, and one or more domains of the receptor is on the cytoplasmic face. The ligand interacts with the receptor to elicit a conformational change in the inside face (cytoplasmic domain) of the receptor- this causes many further steps to take place within the cell.

** The ligand does not enter the cell, but the signal has been transduced across the membrane. The pathway is called signal transduction.

Four classes of cell-surface receptors

1. Ion channel receptors (ligand gated channels)- span plasma membrane and form a channel. Binding of ligand either opens or closes the channel.

2. G protein-coupled receptors (GPCR) - ligand binds the receptor, conformational change in the receptor activates a hetertrimeric G protein. This in turn activates an "effector" molecule (usually an enzyme). The enzyme produces another signal- a small molecule called the 2nd messenger that can diffuse in the cell and interacts with proteins or enzymes to have downstream effects. The ligand is considered the 1st messenger.

3. Receptor tyrosine kinase- has enzymatic activity that is activated by ligand binding. It phosphorylates tyrosines --> activation of monomeric G protein ý downstream target effects.

4. Tyrosine kinase-linked receptors- bind a ligand, but does not have enzymatic activity. The cytoplasmic face undergoes a conformational change and can now bind a tyrosine kinase.

Things to keep in mind

1. The same ligand can elicit different responses in different cells (different cells have different targets).

• adrenaline in heart muscle cells --> increased contraction rate
• adrenaline in smooth muscle cells of the intestine --> decreased contraction rate
• the first few components of these pathways may be the same, but there are different downstream targets.

2. Different ligands can elicit the same response.

3. The multiple steps of transduction pathways allow for different levels for regulation and amplification.

• 1 peptide hormone binds --> many 2nd messenger molecules. Signals can be amplified 100-1000 fold.

G protein-coupled receptors

• largest class of membrane receptors- >1000 have been identified
• each has seven transmembrane domains (protein passes through the plasma membrane seven times). In most cases there is a large loop between the domains that is important for interacting with the heterotrimeric G protein.
• these receptors have a similar 3D structure, but are not all identical
• all function as monomers
• all GPCRs interact with a heterotrimeric GTP-binding protein which acts as a molecular switch.
  • GTP bound --> active
  • GDP bound --> inactive

G proteins

• have intrinsic GTPase activity (catalyze hydrolysis of GTP --> GDP + PI). By doing this they convert themselves into the inactive state.
• hetertrimeric- have three different subunits
• a, b, g
• a always binds GTP or GDP and has GTPase activity
• G protein in its trimeric state (a b g) with a binding GDP is inactive
• When receptor undergoes conformational change and interacts with G protein (a b g), this causes exchange of GTP for GDP. The GDP is removed, and GTP, which is in a higher concentration in the cell, now binds. The subunits dissociate ( a:GTP from bg )
• Either a:GTP or bg can interact with effectors - both are in their active state
• These subunits stay associated with the membrane via lipid modifications- lipid regions are attached to the a and bg to link them covalently to membrane.
• In most cases, the effector resides within the plasma membrane

Fig. 26.11- Activation of heterotrimeric G protein, which can slide laterally on the surface of the bilayer.

Fig. 26.10 - Many types of G a subunits- some activate effectors, others inhibit effectors. Examples of different pathways- could result in increased cAMP, or decreased cGMP. G protein stimulates adenylyl cyclase, which increases level of cAMP (the 2nd messenger) in cell.

G a subunit binds to and activates adenylyl cyclase, which catalyzes ATP --> cAMP + PPI. cAMP is a very potent 2nd messenger. As long as a subunit bound to adenylyl cyclase enzyme, cAMP will increase- amplification (one ligand, many cAMPs). Increased cAMP will have different effects in different cells- handout shows some of the responses.

G a is also a GTPase - so eventually GTP --> GDP + PI is catalyzed. a:GDP can no longer activate adenylyl cyclase- the molecular switch is off. This also promotes reassociation of a with bg.

Effect of cAMP on Protein Kinase A (PKA)

• Increased cAMP activates PKA (a cAMP-dependent protein kinase)
• PKA
• 2 regulatory subunits - each binds cAMP, which causes them to release the
• 2 catalytic subunits, which can now enter the nucleus
• cAMP increases transcription of genes containing a response element called CRE (cAMP response element)
• catalytic subunits phosphorylate CREB protein (cAMP response element binding protein), which activates it as a transcription factor.
• CREB:P interacts with a coactivator at high affinity, stimulating transcription- but only when CREB is phosphorylated

Fig. 26.34 - GPCRs are also called serpentine receptors (have seven transmembrane domains)

Receptor Tyrosine Kinase

• binding of ligand induces the RAS/MAPK phosphorylation cascade.
• one kinase gets activated, which activates another kinase, which activates another kinase, etc. Š until finally a kinase activates a transcription factor. There are many opportunities for amplification because one kinase can activate many substrates.
• respond to growth factors (also called cytokines) that stimulate cell division. Examples - EGF, PDGF.
• Binding of ligand induces dimerization of receptor - now activated, it autophosphorylates. Phosphotyrosines allow binding of other proteins. Downstream of this, have activation of Ras (a monomeric G protein), which activates Raf, then MEK, then MAP kinase, then transcription factors (see handout).
• single transmembrane domain
• most are single polypeptide chains, but there are exceptions (insulin receptor is a dimer)
• ligand binds, causing conformational change on inside which facilitates dimerization of receptors.
• C terminus has kinase activity- each phosphorylates tyrosines on the other receptor, so have Tyr:Ps on each domain. This is called autophosphorylation.
• both receptors have to have kinase activity in order for the receptor to become activated (one receptor is not sufficient).
• These phosphorylated Tyr's serve as docking sites- proteins bind here and in the vicinity.

Targets that bind Tyr:P can be:

1. substrates for RTK
2. enzymes- when phosphorylated, switch into active state. Could be a kinase.
3. adapter proteins- role is to allow RTK to activate another component. They bind to RTK and another protein, and activate it. They are not phosphorylated by a kinase, nor do they have enzymatic activity.

Fig. 26.17 - many different tyrosines phosphorylated- each can be a binding site for a different protein. A scaffold of Tyr:Ps can diverge into multiple transduction pathways. 1 kinase doesn't just bind one kinase or 1 enzyme.

SH2 domain (src homology domain) interacts with Tyr:P, which is conserved in different proteins. SH2 domain recognizes Tyr:P and 3-5 aa's C terminal to it. Different proteins recognize different strings of aa's (specificity). It's a very small binding site, but SH2 domain binds with a high affinity.

Adapter proteins

• involve activation of monomeric G protein Ras
• Ras needs assistance of proteins to cycle (GAP and GEF)
• GAP = GTPase activating protein - an accessory protein that assists the intrinsic GTPase activity of Ras. GAP inactivates Ras.
• GEF = guanine nucleotide exchange factor- facilitates release of GDP from Ras. Now GTP can bind because it is in a higher concentration in the cell. GEF facilitates Ras activation.

RTK indirectly activates Ras

• Grb2 (which has SH2 and SH3 domains) is an adapter protein that binds to Tyr:P, recruits cytosolic SOS to the membrane. SOS is a GEF for Ras.
• Ras is lipid-modified- has a group that inserts into lipid bilayer.
• SOS helps Raf release GDP; Raf binds GTP and becomes activated.

Fig. 26.21 - activation of Ras. SH2 domain of Grb2 binds Tyr:P, SH3 domain binds SOS. The molecular switch is now activated.

Activated Ras --> phosphorylation cascade

• Ras indirectly activates Raf (a Ser/Thr kinase)
• Activated Raf phosphorylates MEK (MAP and ERK kinase). ERK = extracellular response kinase. MEK phosphorylates tyrosines and threonines (unusual).
• MEK phosphorylates MAPK (mitogen-activated protein kinase). A mitogen is a small molecule that stimulates division. Growth factors or cytokines are mitogens.
• Active MAPK phosphorylates transcription factors (now have increased affinity for DNA, coactivator, or some other protein). This generally increases (or sometimes decreases) transcription- regulation of gene expression.
• In some cases, MAPK enters the nucleus; in others, MAPK phosphorylates a transcription factor that goes into the nucleus- has to do with the duration of the signal.

Fig. 26.20 - shows in cartoon form.

Signal transduction pathway --> cell division. Some things that can go wrongŠ

1. Mutation in RTK- no longer requires ligand binding to activate the kinase. Receptor can dimerize and activate without ligand. Result- phosphorylation cascade will be on continuously- kinases --> TFs --> uncontrolled cell division.
2. Mutation in Ras- can bind GTP but not hydrolyze it, even with GAP. Ras:GTP is active, can't cycle into inactive form. The cascade is on and transcription is on, and cell division occurs continuously.
   • a point mutation causing a single aa change can cause a protein not to be regulated in the normal manner

Many signal transduction genes are proto-oncogenes (oncos = tumor or mass). Their products have the ability to transform a eukaryotic cell into a tumor cell.

Can also get mutations that cause an overproduction of a proto-oncogene gene product. wt gene product is made, there is just way too much of it.

• example- overexpression of transcription factor c-myc causes Burkitt's lymphoma. A translocation (break and rearrangement in chromosome) puts myc gene in immunoglobulin gene cluster (B cells). Active promoter drives production of myc in B cells.
• myc is a substrate for MAPK. MAPK phosphorylates myc, which causes increased transcription.

Fig. 28.14 - many oncogenes code for components of signal transduction cascades.

Fig. 26.29 - different types of signal transduction pathways can converge to cause an increase in one cellular protein with a downstream target.

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