MOLECULAR CELL BIOLOGYSIXTH EDITION
Copyright 2008 © W. H. Freeman and Company
CHAPTER 15Cell Signaling I:
Signal Transduction and Short-Term Cellular Responses
Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira
Characterizing Cell-Surface Receptors
ü Receptors bind ligands with considerable Specificity, determined by noncovalent interactions between a ligand and specific aa in the receptor (Fig 15-3).
ü The concentration of ligand at which half its receptors are occupied, the Kd, can he determined experimentally and is a measure of the affinity of the receptor for the ligand (Fig 15-4).
ü The maximal response of a cell to a particular ligand generally occurs at ligand concentrations at which most of its receptors are still not occupied (Fig 15-6).
3
ü Because the amount of a particular receptor expressed is generally quite low (ranging from =1000 to 50,000 molecules per cell), biochemical purification may not be feasible.
ü Genes encoding low-abundance receptors for specific ligands often can be isolated from cDNA libraries transfected into cultured cells.
ü Functional expression assays can determine if a cDNA encodes a particular receptor and are useful in studying the effects on receptor function of specific mutations in its sequence (see Figure 15-7).
4
15.3 Highly Conserved Components of Intracellular Signal-Transduction Pathways
External signals induce two major types of cellular responses:(1 ) Changes in the activity or function of specific enzymes and other proteins that pre-exist in the cell
(2) Changes in the amounts of specific proteins produced by a cell, most commonly by modification of transcription factors that stimulate or repress gene expression (see Figure 15-1).
5
Ø ln general, the first cycle of response occurs more rapidly
than the second.
Ø Signalling from G protein-coupled receptors, often results
in changes in the activity of pre-existing proteins.
Ø Although activation of these receptors on some cells also
induces changes in gene expression.
6
Ø Other classes of receptors operate primarily but not
exclusively to modulate gene expression.
Ø Transcription factors activated in the cytosol by these
pathways move into the nucleus, where they stimulate (or
occasionally repress) transcription of specific target
genes.
Ø We will discuss this in the next Chapter
7
q Several intracellular proteins or small molecules are employed in a
variety of signal-transduction pathways.
q These include cytosolic enzymes that add or remove phosphate groups
from specific target proteins.
q Ligand binding to a receptor activates or inhibits these enzymes, whose
action in turn activates or inhibits the function of their target proteins.
q G proteins, another component of many signal-transduction pathways,
shuttle between a state with a bound GTP that is capable of activating
other proteins and a state with a bound GDP that is inactive.
8
q A number of small molecules (e.g., Ca2+ and cyclic-AMP) are also frequently used in intracellular signal-transduction pathways.
q A rise in the concentration of one of these molecules results in its binding to an intracellular target protein, causing a conformational change in the protein that modulates its function.
q Here we review the basic properties of these intracellularsignal-transducing molecules.
9
GTP-Binding Proteins Are Frequently Used As On/Off Switches
The proteins in the GTPase superfamily (guanine nucleotide-binding proteins) are: i. Turned "on" when they bind GTP and ii. Turned "off" when the GTP is hydrolysed to GDP(Please see figure 3-32, Lodish).
10
11
v Signal-induced conversion of the inactive to active state is mediated by a guanine nucleotide- exchange factor (GEF)
v GEF causes release of GDP from the switch protein v Subsequent binding of GTP, favoured by its high intracellular
concentration relative to its binding affinityv Induces a conformational change in at least two highly
conserved segments of the protein, termed switch I and switch II, allow binding
v Activate other downstream signalling proteins (Figure lS-8).
12
Switching mechanism for G proteins.
13
14
Ø The intrinsic GTPase activity of the protein then hydrolyses the bound GTP to GDP and P.
Ø Thus changing the conformation of switch l and switch II from the active form back to the inactive form.
Ø The rate of GTP hydrolysis regulates the length of time the switch protein remains in the active conformation and able to signal downstream.
Ø The slower the rate of GTP hydrolysis, the longer the protein remains in the active state.
15
q The rate of GTP hydrolysis is often modulated by otherproteins.
q For instance, both GTPase activating proteins (CAP) and regulator of G protein signalling (RGS) proteins accelerate GTP hydrolysis.
q Many regulators of G protein activity are themselves controlled by extracellular signals.
16
✑ Two large classes of GTPase switch proteins are used in signalling.
v Trimeric (large) G proteins, directly bind to and are activated by certain cell-surface receptors. The activated receptor functions as a GEF and triggers release of GDP and binding of GTP.
v Monomeric (small) G-proteins, such as Ras and various Ras-like proteins, play crucial roles in many pathways that regulate cell division and cell motility
17
v These G proteins frequently undergo activating mutations in cancers. Ras is linked indirectly to receptors via adapter proteins (Chap 16)
Ø All G switch proteins contain regions like switch I and switch llthat modulate the activity of specific effector proteins.
Ø This is done by direct protein-protein interactions when the G protein is bound to GTP.
Ø Despite these similarities, the two classes of GTP-binding proteins are regulated in very different ways.
18
Protein Kinases and Phosphatases are Employed in Virtually All
Signalling Pathways
Ø Activation of virtually all cell-surface receptors leads directlyor indirectly to changes in protein phosphorylation through the activation of :
1. Protein kinases, which add phosphate groups to specific residues,
1. protein phosphatases, which remove phosphate groups from a specific residue.
19
Animal cells contain two types of protein kinases:
1. Those that add phosphate to the hydroxyl group on tyrosine residues (Tyrosine Kinases)
2. Those that add phosphate to the hydroxyl group on serineor threonine (or both) residues (Serine/Threonine Kinases)
20
v Phosphatases can act in concert with kinases to switch the function of various proteins on or off.
v The human genome encodes about 600 protein kinases and 100different phosphatases.
Ø In some signalling pathways, the receptor itself possessesintrinsic kinase or phosphatase activity;
Ø in other pathways, the receptor interacts with cytosolic or membrane-associated kinases.
21
✑ Importantly the activity of all kinases is highly regulated. ü Commonly the catalytic activity of a protein kinase itself
is modulated by phosphorylation by other kinases, via a. Direct binding to other proteinsb. Changes in the levels of various small intracellular
signalling molecules.
ü The resulting cascades of kinase activity are a commonfeature of many signalling pathways.
22
✑ In general, each protein kinase phosphorylates specific residues in a
set of target proteins whose patterns of expression generally differ
in different cell types.
✑ Many proteins are substrates for multiple kinases each of which
phosphorylates different amino acids.
✑ Each phosphorylation event can modify the activity of a particular
target protein in different ways,
✑ Some activating its function, others inhibiting it.
23
Ø Example: glycogen phosphorylase kinase, a key regulatory enzyme in glucose metabolism.
Ø The activity of a protein kinases is opposed by the activity of protein phosphatases. (Activation vs.inactivation)
v Thus the activity of a protein in a cell can be a complexfunction of the activities of the usually multiple kinasesand phosphatases that act on it.
24
Top Related