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).

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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).

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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).

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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.

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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

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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.

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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.

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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).

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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).

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Switching mechanism for G proteins.

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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.

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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.

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✑ 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

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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.

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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.

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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)

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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.

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✑ 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.

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✑ 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.

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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.

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