Chapter 25: Enzyme-Coupled Receptors:Signaling Through Receptor Tyrosine Kinases

Question 1

Enzyme-Coupled Receptors and enzymes

Answer 1

Are either enymes or are very closely associated with enzymes

Question 2

Enzyme-coupled receptors

Answer 2

•Enzyme-coupled receptors differ from G protein-linked receptors in two fundamental ways:

• have only one transmembrane domain per receptor monomer
• do not interact with G proteins, but instead use enzymatic activity to transduce the signal of ligand binding
• Most of the receptors in this class are protein kinases; however, some receptors have protein phosphatase or guanylyl cyclase activity
• In most cases, activation of enzyme activity occurs in response to dimerization of receptor monomers upon ligand binding

Question 3

Receptor tyrosine kinases (RTKs) Definition

Answer 3

Phosphorylate on specific tyrosines

Question 4

Cytokine receptors (tyrosine kinase-associated receptors)

Answer 4

Associate with intracellular tyrosine kinases

Question 5

Receptor serine/threonine kinases

Answer 5

Phosphorylate specific serines or threonines

Question 6

Receptor guanylyl cyclases

Answer 6

Synthesize cGMP

Question 7

Receptor tyrosine phosphatases

Answer 7

Remove phosphoryls from tyrosines

Question 8

Receptor Tyrosine Kinases (RTKs)

Answer 8

• Most abundant type of enzyme-coupled receptor, with about 60 members classified into 20 structural families
• In all cases, binding of a ligand activates the cytosolic internal tyrosine kinase domain
• RTKs regulate cell proliferation, cell growth, cell differentiation, cell migration, and, during development, cell fate
• RTKs were discovered by studying a class of signal molecues known as growth factors (e.g. Epidermal Growth Factor=EGF, Fibroblast Growth Factor=FGF)
• Growth factors are signal molecules that act as local mediators to stimulate growth and proliferation of specific cell types

Question 9

Her2/Neu (EGF) Receptor

Answer 9

RTKs typically span the plasma membrane once and are monomers in the absence of ligand.

Question 10

Dimerization of RTKs causes activation of the Tyrosine Kinase Acftivity by 2 Mechanisms, Depending on the Type of RTK

Answer 10

1. Cross phosphorylation=autophosphorylation: the activated tyr kinase domains phosphorylate each other.
2. Conformational changes in the domains in response to ligand binding end up having one tyr kinase domain (activator) activationg the other the tyr kinase domain (receiver), and the reciver then phophorylates both subunits

Question 11

Once the internal domains of RTK are Phosphorylated, They Serve as Docking sites for Signaling Proteins

Answer 11

1. The ligand can be a dimer itself and thus bind to the 2 receptros simultaneously
2. A monomeric ligand can bind to 2 receptors simultaneously to bring them together
3. 2 ligands can bind independently to 2 receptors to bring them together
4. Some receptors like the insulin receptor are already a dimer, and the ligand just induces a conformational that activates the kinase activity

Question 12

Phosphotyrosines Serve as Recognition (Docking) Sites for…

Answer 12

Signaling MOlecules/Enzymes

Note: Different SH2- or PTB-containing proteins recognize different phosphotyrosine contexts

Question 13

Some Proteins that are Recruited to the Activated RTK Serve as “Adaptors”

Answer 13

• Some proteins recruited to activated RTKs have no enzymatic activity and instead act as adaptor (or bridging) proteins that link intracellular signaling proteins together
• One of the first adaptor proteins, which was identified by its ability to bind to RTKs, was Grb2. Grb2 links the MAP kinase cascade to RTKs in some cases
• Grb2 is composed of little more than a central SH2 domain surrounded by 2 SH3 domains
• SH3 domain (src homology domain 3) is a dinstinct modular interaction domain that recognizes proline-rich peptides 9-10 residues long in proteins

Question 14

Adaptor Proteins

Answer 14

Link or Bridge 2 Signaling Proteins Together Through Interaction Domains such as SH3 Domains

Question 15

RTK’s are Proto-Oncogenes and Often Become Oncogenic

Answer 15

The RTK can become mutated so that it dimerizes and becomes constitutively active without the ligand, or the external domain can be truncated, whic also activates the receptor without ligand.

Question 16

Mutation of the Her2 and EGF Receptors Leads to…

Answer 16

Activation without ligand

Both receptors bind EGF

Her2/Neu is often mutated or overexpressed in “hormone-independent” breast cancer (BCa)

Question 17

Herceptin

Answer 17

Is an antagonist

INterferes with EGF signaling through the Her2/Neu receptor

Question 18

Major Pathways Activated by RTKs

Answer 18

Question 19

Src Protein Kinase

Answer 19

• Src was the first protein kinase discovered because it was part of the Rous sarcoma virus
• It is a member of a subfamily of 9 similar protein kinases that are only found in multicelllular animals
• These are all cytosolic tyrosine kinases, although covalent binding to a fatty acid attaches them to the plasma membrane

Question 20

Summary of Signaling Through the Src Tyrosine Kinase

Answer 20

AFter Src is activated, it phosphorylates the RTK on other sites, which augments receptor kinase activity and creates additional SH2 recruitment sites. This allows the binding of Grb2-Sos and PI 3-kinase. In other words, activation of pathways is sequential.

Question 21

Src as an Oncoprotein

Answer 21

• Src is a key protein in promoting cell survival, cell proliferation, cell migration, and angiogenensis
• These are all hallmarks of cancer, especially metastatic cancer, and 50% of tumors from colon, liver, lung, breast and the pancreas have abnormaly activated Src.
• A number of small molecules ares in clinical use and others are in clinical trials that inhibit Src. These include competitive inhibitors of the ATP-binding site and of the substrate binding site

Question 22

MAP Kinase Cascade

Answer 22

Mitogen Activated Protein Kinase

Question 23

Mitogens

Answer 23

Signaling molecules that induce cell proliferationi such as growth factors, estrogen and testosterone

Question 24

Ras

Answer 24

• Key component of MAP kinase cascade
• All of the Ras family members are “small” G proteins (170 aa) or monomeric G proteins to distinguish from the large G-alpha (300 aa) proteins associated with G protein-coupled receptors
• All of the Ras family members function as binary switches

Question 25

GTPase Activating Proteins (GAPs)

Answer 25

Reduce Ras activation by increasing its ability to hydrolyze GTP (~100-fold). Keep about 95% of Ras inactive in the absence of a signaling molecule. Usually called Ras-GAPs

Question 26

Guanine Exchange Factors (GEFs)

Answer 26

Accelerate Ras activaton by promoting the exchange of GTP for the bound GDP

Ex: SOS

Question 27

The Importance of Being Turned Off

Answer 27

• The intrinsic GTPase activity of Ras is weak and only hydrolyzes GTP slowly
• GTPase Activating Proteins (GAPs, Ras-GAPs) act to enhance the weak GTPase activity of monomeric G proteins and promote conversion to the inactive state
• All oncogenic forms of Ras analyzed from human cancers have mutations that interfere with the ability of Ras to interact with Ras-GAP
• These oncogenic forms of Ras are mostly in the GTP bound (active) state and continually signal to cell to promote cell division

Question 28

Activation of Ras by an RTK

Answer 28

Grb2 recognizes a specific phosphorylated tyrosine on the RTK by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates guanine nucleotide exchange, which activates Ras.

Question 29

Activation of Ras is Transient

Answer 29

FRET (fluorescence resonance energy transfer) analysis demonstrated that the activation of Ras is very transient. To do this, yellow fluorescent protein (YFP) was incorporated into RAS, and GTP was linked to a red fluorescent dye and injected into cells. When RAS binds GTP, the energy emitted by YFP activates the red dye and that emits fluorescent light, which can be detected and measured. The results indicate that some activated Ras can be detected by 30 sec after EGF addition, the peak of activation is about 3 min, and all Ras is inactive by 6 min.

Question 30

MAP Kinase Casade

Answer 30

Question 31

MAP Kinase Cascad Linked to an RTK

Answer 31

Phosphorylation of Myc, Jun, & Fos leads to the transcription of genes involved in cell proliferation.

Question 32

Notes on MAP kinase Cascade

Answer 32

• GTP-bound Ras interacts directly with the ser/thr-specific protein kinase Raf (a MAP kinase kinase kinase) to activate it
• Raf was first discovered as an oncogene; oncogenic forms of Raf do not require signals from Ras, Ras-GEF or RTK to promote cell division. (~75% of melanomas are caused by Raf)
• Raf only has one catalytic substrate - the protein kinase Mek (a MAP kinase kinase)
• Mek is unusual in that it contains both ser/thr protein kinase activity and tyrosine protein kinase activity (on separate structural domains)
• Activated Mek only has one catalytic substrate family, MAP kinases, most commonly the protein kinase Erk
• Activation of MAP kinase requires phosphorylation of a threonine and a tyrosine that are separated by only a single amino acid
• MAP kinases have many targets

Question 33

Activated MAP Kinase can act on target proteins in both the cytoplasm and the nucleus

Answer 33

One action of MAP kinase is to activate gene transcription of genes involved in cell proliferation by phosphorylating and activating transcription activators

Question 34

Different Signals Activate Different MAPK Pathways

Answer 34

Question 35

Mechanisms for Attenuation and Termination of RTK Activation

Answer 35

1) Antagonists (Herceptin®)
2) Phosphorylation or dephosphorylation

3) Receptor sequestration (endocytosis)

4) Receptor internalization and degradation
5) Receptor inhibition