Intracellular Signalling Flashcards
How do RTKs exist in the absence of extracellular signalls?
- In the absence of extracellular signals, most RTKs exist as monomers in which the internal kinase domain is inactive. Binding of ligand brings two monomers together to form a dimer.
- In most cases, the close proximity in the dimer leads the two kinase domains to phosphorylate each other, which has two effects. First, phosphorylation at some tyrosines in the kinase domains promotes the complete activation of the domains. Second, phosphorylation at tyrosines in other parts of the receptors generates docking sites for intracellular signaling proteins, resulting in the formation of large signaling complexes that can then broadcast signals along multiple signaling pathways. Mechanisms of dimerization vary widely among different RTK family members. In some cases, as shown here, the ligand itself is a dimer and brings two receptors together by binding them simultaneously. In other cases, a monomeric ligand can interact with two receptors simultaneously to bring them together, or two ligands can bind independently on two receptors to promote dimerization.
- In some RTKs—notably those in the insulin receptor family—the receptor is always a dimer , and ligand binding causes a conformational change that brings the two internal kinase domains closer together.
- Although many RTKs are activated by transautophosphorylation as shown here, there are some important exceptions, including the EGF receptor
What are SH2 Domains?
- SH2 Domain: A polypeptide segment of about 100 amino acid residues in length that binds phosphotyrosine when the phospho- tyrosine is present within a preferred amino acid sequence.
- The purpose of SH2 domains is to mediate protein-protein interactions.
- The name SH2 is derived from Src homology region 2 .
What is Src?
Src is a NON-RECEPTOR tyrosine kinase.
If you align Src with other proteins involved in signal transduction, common domains are easily spotted.
- SH1 is the kinase domain
- SH2 binds phosphotyrosine
- SH3 binds proline rich sequences (9-12aa)
Describe SH2 and SH3 Domains in the Activation of Src
(A) The SH2 group of Src (blue) normally binds in an intramolecular fashion to a phosphotyrosine residue (pY; red) at position 527 near the C-terminus of Src. This binding causes the catalytic cleft of Src, which is located between the N-lobe and the C-lobe of the kinase domain, to be obstructed. At the same time, the SH3 (light green) domain binds a proline-rich portion in the linker segment (magenta) between the N-lobe of the kinase domain and the SH2 domain.
(B) However, when a domain of the PDGF receptor (indicated here as a “Src activator”) becomes phosphorylated, one of its phosphotyrosines (red) serves as a ligand for this SH2 group of Src, causing the SH2 to switch from intramolecular binding to intermolecular binding. Concomitantly, the SH3 group of Src detaches from its intramolecular binding to bind a prolinerich domain (PXXP) on the receptor. These changes open up the catalytic cleft of Src, placing it in a configuration that enables it to fire.
(C) A final phosphorylation of tyrosine 416 moves an obstructing oligopeptide activation loop (dark green) out of the way of the catalytic cleft, yielding the full tyrosine kinase activity of Src.
Explain the The Domain Structure of Src
Sequence analyses of the Src protein revealed three distinct structural domains that are also found in a large number of other signaling proteins.
- (A) In this ribbon diagram of Src—the product of X-ray crystallography—coils indicate α-helices, while flattened ribbons indicate β-pleated sheets. Like many other subsequently analyzed protein kinases (for example, see Figure 5.17), the catalytic SH1 domain is seen to be composed of two subdomains (right), termed the N- and C-lobes of the kinase (yellow, orange). Between these lobes is the ATP-binding site where catalysis takes place (red stick figure). The SH2 and SH3 domains (blue, light green; left) are involved in substrate recognition and regulation of catalytic activity. Connecting sequences are shown in dark green.
- (B) A space-filling model of Src is presented in the same color scheme.
Show SH2
Domains
- (A) This drawing of a receptor for platelet-derived growth factor (PDGF) shows five phosphotyrosine docking sites, three in the kinase insert region and two on the C-terminal tail, to which the three signaling proteins shown bind as indicated. The numbers on the right indicate the positions of the tyrosines in the polypeptide chain. These binding sites have been identified by using recombinant DNA technology to mutate specific tyrosines in the receptor. Mutation of tyrosines 1009 and 1021, for example, prevents the binding and activation of PLCγ, so that receptor activation no longer stimulates the inositol phospholipid signaling pathway. The locations of the SH2 (red) and SH3 (blue) domains in the three signaling proteins are indicated. (Additional phosphotyrosine docking sites on this receptor are not shown, including those that bind the cytoplasmic tyrosine kinase Src and two adaptor proteins.) It is unclear how many signaling proteins can bind simultaneously to a single RTK.
- (B) The three-dimensional structure of an SH2 domain, as determined by x-ray crystallography. The binding pocket for phosphotyrosine is shaded in orange on the right, and a pocket for binding a specific amino acid side chain (isoleucine, in this case) is shaded in yellow on the left. The RTK polypeptide segment that binds the SH2 domain is shown in yellow (see also Figure 3^10).
- (C) The SH2 domain is a compact, “plug-in” module, which can be inserted almost anywhere in a protein without disturbing the protein’s folding or function (discussed in Chapter 3). Because each domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences
SH2 Domain Binding
a Phosphopeptide
The phosotyrosine and the +3 isoleucine fit into pockets of the SH2-domain.
SH2 Domain-Containing Proteins Bind to Phosphorylated Receptors
- (A) This schematic diagram of a molecule of the platelet-derived growth factor-β (PDGF-β) receptor, which omits its tyrosine kinase domain, reveals a large number of tyrosine residues in its C-terminal domain that undergo phosphorylation following ligand binding and receptor activation. (The positions of these tyrosine residues in the receptor polypeptide chain are indicated by the numbers.) Listed to the left are seven distinct cytoplasmic proteins, each of which can bind via its SH2 domain(s) to one or more of the phosphotyrosines of the PDGF-β receptor. The three amino acid residues (denoted by the single-letter code) that flank each tyrosine (Y) residue on its C-terminal side define the unique binding site recognized by the various SH2 domains of these seven associated proteins.
- (B) Similarly, a constellation of phosphotyrosines can be formed after the EGF receptor binds its ligand, often forming heterodimers with the HER2 receptor protein. However, the spectrum of SH2-containing proteins that associates with the EGF-R is quite different, allowing this receptor to activate a different set of downstream signaling pathways from that of the PDGF-β receptor.
TRUE or FALSE: The recruitment of SH2 domain-containing proteins is essential to most classes of single pass transmembrane receptors (not just RTK)
TRUE
What are the consequences of Binding the SH2 Domain-Containing Protein?
- translocate the SH2 domain-containing protein to the plasma membrane
- induce the proximity of the SH2 domain- containing protein to other signaling proteins
- facilitate direct phosphorylation of the SH2 domain-containing protein by the receptor
What is Grb2?
- Grb2 is an adaptor. Grb2 acts as a link between tyrosine phosphorylated proteins and proteins that display the proline rich sequences recognized by SH3 domains.
- Grb2 has NO intrinsic catalytic activity. Grb2 is highly conserved (Grb2=Sem5=Drk).
Show the Structure of Grb2
The SH2 Domain of Grb2 Recruits Grb2 to The Phosphorylated Receptor
SH3 Domain with Prolines Bound
The SH3 domain (ribbon diagram), also shown in a different orientation from that of Figure 6.7, recognizes as its ligands proline-rich oligopeptide (blue stick figure) sequences in partner proteins
What do SH3 Domains bind to?
SH3 domains bind to linear proline-rich sequences
Describe how RTK Signalling Uses Grb2, Sos and Ras?
- Grb2 recognizes a specific phosphorylated tyrosine on the activated receptor by means of an SH2 domain and recruits Sos by means of two SH3 domains.
- Sos stimulates the inactive Ras protein to replace its bound GDP by GTP, which activates Ras to relay the signal downstream.
Describe the Activation of
Monomeric G Protein
- GTPase-activating proteins (GAPs) inactivate the protein by stimulating it to hydrolyze its bound GTP to GDP, which remains tightly bound to the inactivated GTPase.
- Guanine nucleotide exchange factors (GEFs) activate the inactive protein by stimulating it to release its GDP; because the concentration of GTP in the cytosol is 10 times greater than the concentration of GDP, the protein rapidly binds GTP and is thereby activated.
Sos Binding Promotes GTP Binding
Switch I and Switch II change conformation depending on whether GDP or GTP are bound.
FRET Can Be Used to Study the Kinetics of Ras activation
A) Schematic drawing of the experimental strategy. Cells of a human cancer cell line are genetically engineered to express a Ras protein that is covalently linked to yellow fluorescent protein (YFP). GTP that is labeled with a red fluorescent dye is microinjected into some of the cells. The cells are then stimulated with the extracellular signal protein EGF, and single fluorescent molecules of Ras-YFP at the inner surface of the plasma membrane are followed by video fluorescence microscopy in individual cells. When a fluorescent Ras-YFP molecule becomes activated, it exchanges unlabeled GDP for fluorescently labeled GTP; the yellow-green light emitted from the YFP now activates the fluorescent GTP to emit red light (called fluorescence resonance energy transfer, or FRET; see Figure 9–26). Thus, the activation of single Ras molecules can be followed by the emission of red fluorescence from a previously yellow-green fluorescent spot at the plasma membrane.
As shown in (B), activated Ras molecules can be detected after about 30 seconds of EGF stimulation. The red signal peaks at about 3 minutes and then decreases to baseline by 6 minutes. As Ras-GAP is found to be recruited to the same spots at the plasma membrane as Ras, it presumably plays a major part in rapidly shutting off the Ras signal
Peak Ras Activity Is after a Few Minutes
- Activated Ras molecules can be detected after about 30 seconds of EGF stimulation.
- The red signal peaks at about 3 minutes and then decreases to baseline by 6 minutes.
- As Ras-GAP is found to be recruited to the same spots at the plasma membrane as Ras, it presumably plays a major part in rapidly shutting off the Ras signal
