Biochemistry, 4th Edition P106 docx

Binding site for GTP-binding guanylyl cyclase activity Channel opens and closes in response to extracellular ligand binding G-protein–coupled receptor N C Receptor tyrosine kinase or rec

32.3 How Do Signal-Transducing Receptors Respond

to the Hormonal Message?

All receptors that mediate transmembrane signaling processes fit into one of three

receptor superfamilies(Figure 32.7):

1 The G-protein–coupled receptors (see Section 32.4) are integral membrane

pro-teins with an extracellular recognition site for ligands and an intracellular

recog-nition site for a GTP-binding protein (see following discussion).

2 The single-transmembrane segment (1-TMS) catalytic receptors are proteins with

only a single transmembrane segment and substantial globular domains on both

the extracellular and the intracellular faces of the membrane The extracellular

domain in the ligand recognition site and the intracellular catalytic domain is

ei-ther a tyrosine kinase or a guanylyl cyclase.

3 Oligomeric ion channels consist of associations of protein subunits, each of

which contains several transmembrane segments These oligomeric structures

PDZ (dimer)

Pumilio, complexed with RNA (green)

FIGURE 32.5 Five of the protein modules found in cell signaling proteins The binding specificity of these

mod-ules is shown in Figure 32.6a SH3 domains bind to proline-rich peptides; SH2 domains bind to phosphorylated

tyrosine residues; PH domains bind to phosphoinositides (such as IP 3 ); PDZ domains bind to the terminal four

or five residues of a target protein; pumilio domains bind to segments of RNA A given protein may have a

number of these protein modules, giving it the ability to interact with multiple partners.

SH3

(a)

(b)

IP3

B

D C

COO ⴚ

N

XXX XXSXX

XXYXX

PH

IP 3

PDZ

SH3

P X X P

COO ⴚ

XXX

P P

A

FIGURE 32.6 (a) Many signaling proteins consist of

com-binations of protein modules, each with a specific

bind-ing or enzymatic function (b) Such multifunctional

pro-teins can act as scaffolds that direct the assembly of

large signaling complexes, termed signalsomes.

Binding site for GTP-binding

guanylyl cyclase activity

Channel opens and closes in response to extracellular ligand binding

G-protein–coupled receptor N

C

Receptor tyrosine kinase

or receptor guanylyl cyclase

Oligomeric ion channels

FIGURE 32.7 The membrane receptor superfamilies The

G-protein–coupled receptors are named for the

GTP-binding proteins that mediate some of their effects The

receptor tyrosine kinases and receptor guanylyl cyclases

contain intracellular enzymatic domains that respond to

extracellular hormone binding The oligomeric ion

chan-nels (some of which were discussed in Chapter 9) open

and close in response to ligand binding and/or changes

of the transmembrane electrochemical potential.

are ligand-gated ion channels Binding of the specific ligand typically opens the

ion channel The ligands for these ion channels are neurotransmitters

The G-Protein–Coupled Receptors Are 7-TMS Integral

Membrane Proteins

The G-protein–coupled receptors (GPCRs) have primary and secondary structure

similar to that of bacteriorhodopsin (see Chapter 9), with seven transmembrane

-helical segments; they are thus known as 7-transmembrane segment (7-TMS)

pro-teins Rhodopsin and the -adrenergic receptors, for which epinephrine is a ligand,

are good examples (Figure 32.8) The site for binding of cationic catecholamines

to the adrenergic receptors is located within the hydrophobic core of the receptor

Binding of hormone to a GPCR induces a conformation change that activates a

GTP-binding protein, also known as a G protein (discussed in Section 32.4)

Acti-vated G proteins trigger a variety of cellular effects, including activation of adenylyl

and guanylyl cyclases (which produce cAMP and cGMP from ATP and GTP),

activation of phospholipases (which produce second messengers from

phospho-lipids) and activation of Ca2and Kchannels (which leads to elevation of

intra-cellular [Ca2] and [K]) (All of these effects are described in Section 32.4.)

The Single TMS Receptors Are Guanylyl Cyclases or Tyrosine Kinases

Receptor proteins that span the plasma membrane with a single helical

trans-membrane segment possess an external ligand recognition site and an internal

do-main with enzyme activity—either receptor tyrosine kinase (RTK) or receptor

Membrane Outside

2 -Adrenergic receptor

Inside

NH3

COO–

Asp 113

FIGURE 32.8 (a) The arrangement of the 2 -adrenergic receptor in the membrane Substitution of Asp 113 in the

third hydrophobic domain of the -adrenergic receptor with an Asn or Gln by site-directed mutagenesis results

in a dramatic decrease in affinity of the receptor for both agonists and antagonists Significantly, this Asp residue

is conserved in all other GPCRs that bind biogenic amines but is absent in receptors whose ligands are not

amines Asp 113appears to be the counterion of the amine moiety of adrenergic ligands (b) The structure of a 2

-adrenergic receptor (pdb id  2RH1).The flexible third intracellular loop and C-terminal segment are not shown.

guanylyl cyclase (RGC) Each of these enzyme activities is manifested in two dif-ferent cellular forms Thus, guanylyl cyclase activity is found in both membrane-bound receptors and in soluble, cytoplasmic proteins Tyrosine kinase activity, on the other hand, is exhibited by two different types of membrane proteins: The RTKs are integral transmembrane proteins, whereas the non-RTKs are peripheral, lipid-anchored proteins

RTKs and RGCs Are Membrane-Associated Allosteric Enzymes

The binding of polypeptide hormones and growth factors to RTKs and RGCs acti-vates the intracellular enzyme activity of these proteins These catalytic receptors are composed of three domains (Figure 32.9): an extracellular receptor-binding domain (which may itself include several subdomains), a transmembrane domain consisting

of a single transmembrane -helix, and an intracellular domain This intracellular

portion includes a tyrosine kinase or guanylyl cyclase domain that mediates the bio-logical response to the hormone or growth factor via its catalytic activity and a regu-latory domain that contains multiple phosphorylation sites The human genome contains at least 58 different RTKs, which can be grouped into about 20 families on the basis of their kinase domain sequences and the various extracellular subdomains

The epidermal growth factor (EGF) receptor and the insulin receptor are

represen-tative of this class of receptor proteins

Given that the extracellular and intracellular domains of RTKs and RGCs are joined

by only a single transmembrane helical segment, how does extracellular hormone binding activate intracellular enzyme activity? How is the signal transduced? As shown

in Figure 32.10, signal transduction occurs by hormone-induced oligomeric

associa-tion of receptors Hormone binding triggers a conformaassocia-tional change in the extracel-lular domain, which induces oligomeric association Oligomeric association allows ad-jacent cytoplasmic domains to interact, leading to phosphorylation of the cytoplasmic

domains and stimulation of cytoplasmic enzyme activity By virtue of these

ligand-Outside

Inside

N

I

II

III

IV

C

Fn3

Fn2b

Fn2a Fn1

Tyrosine kinase

Tyrosine kinase

L2 CR L1

Fn3 Fn2b

Hormone-binding domain

Juxta-membrane domain

Protein kinase–like domain

Protein kinase domains Guanylyl

cyclase

ANP-R

(d)

FIGURE 32.9 (a) The EGF receptor and (b) the insulin

re-ceptor are rere-ceptor tyrosine kinases EGF rere-ceptors are

activated by ligand-induced dimerization, whereas the

insulin receptor is a glycoprotein composed of two

kinds of subunits in an 22 tetramer stabilized by

disul-fide bonds The extracellular portions of the EGF and

in-sulin receptors consist of multiple modules or domains.

Fn refers to a series of FnIII-type domains numbered 1,

2a, 2b, and 3 (c) The atrial natriuretic peptide receptor is

a receptor guanylyl cyclase with a large extracellular

hormone-binding domain and two intracellular

do-mains, and activation typically involves ligand-induced

dimerization (d) The growth factor receptor Ret is a

re-ceptor tyrosine kinase that mediates the effects of

neu-ronal growth factors known as neurotrophins The Ret

receptor domains (yellow) requires a GPI-anchored

coreceptor (blue) for binding of ligands (pink).

induced conformation changes and oligomeric interactions, RTKs and RGCs are

membrane-associated allosteric enzymes.

EGF Receptor Is Activated by Ligand-Induced Dimerization

Human EGF is a 53-amino acid peptide that stimulates proliferation of epithelial

cells (cells that cover a surface or line a cavity in biological tissues) The human EGF

receptor is a 1186–amino acid RTK The extracellular domain, where EGF binds,

contains 622 amino acids and is divided into four domains (Figure 32.9) Domains

I and III are similar -helical barrels, whereas domains II and IV are long, slender,

cysteine-rich domains stabilized by disulfide bonds Domain II is characterized by a

-hairpin structure that protrudes from the middle of the domain (Figure 32.11a)

Prior to EGF binding, EGF receptors exist as inactive monomers in the plasma

membrane In this state, the four domains are folded so that domains II and IV lie

parallel, with the -hairpin of domain II in contact with domain IV (Figure 32.11a).

Binding of EGF to domain I induces a conformation change that rotates domains I

and II so that the bound EGF is brought into contact with domain III and the

-hairpin is extended away from the rest of the structure Pairs of such receptor

monomers then dimerize by mutual association of the -hairpin structures (Figure

32.11b) The next event is the critical step in transmembrane signal transduction

Dimer-ization of the extracellular domains brings the intracellular domains together,

acti-vating the tyrosine kinase activity Thus, EGF-induced dimerization allows an

extra-cellular signal (EGF) to exert an intraextra-cellular response

EGF Receptor Activation Forms an Asymmetric Tyrosine Kinase Dimer

The tyrosine kinase domain of the EGF receptor consists of an N-terminal domain

built around a twisted -sheet and a C-terminal domain that is primarily -helical

(Figure 32.12a) In the inactive, monomeric EGF receptor, the active site of the

tyrosine kinase domain is blocked by a 30-residue loop of the protein (residues

831–860) that is termed the activation loop Extracellular dimer formation by the

Ligand

(a)

(a)

(d)

S S

S

Ligand

(b)

S

ANIMATED FIGURE 32.10 Ligand-induced oligomerization of

membrane receptors can occur in several ways (a) EGF receptor dimerization

and activation involves binding of hormone to the extracellular domains of two

receptor molecules (b) The insulin receptor is a preexisting tetramer Two insulin-binding sites are created by association of the extracellular domains (c) RGCs bind

a single hormone ligand at the dimer interface (Ligand dimers could presumably

act in a similar manner) (d) Neurotrophin activation of Ret involves association of

two molecules of the Ret kinase and two molecules of a GPI-anchored coreceptor

protein See this figure animated at www.cengage.com/login.

Ligand monomer

or dimer

(c) (b)

EGF receptor appears to promote formation of an asymmetric dimer of the intra-cellular tyrosine kinase domains (Figure 32.12b), with the C-terminal lobe of one kinase domain juxtaposed with the N-terminal lobe of the other kinase domain In this asymmetric dimer, one monomer is inactive but acts as an activator of kinase ac-tivity in the other monomer Conversion of the kinase domain from its inactive state

to the active conformation involves rotation of the activation loop out of the active site, making room for a peptide substrate to enter the site (Figure 32.12c)

The activated tyrosine kinase domain of the EGF receptor can phosphorylate several tyrosine residues at its own C-terminus (Figure 32.13), a process termed

autophosphorylation.These phosphorylated tyrosines are binding sites for a variety

of other signaling proteins that contain phosphotyrosine-binding SH2 domains (see Figures 32.5 and 32.6) Each of these SH2-domain–containing proteins can initiate several signal transduction cascades

Binding site for EGF

on domain III

(a)

(b)

Domain I/II rigid body

1

1

EGF

2

2 E

3

3 4

4

E

1 2 2 1

4 4

Dimer Auto-inhibited

II III

IV

I

I

III

II

IV

FIGURE 32.11 (a) In the absence of hormone, the extracellular domain of the EGF

recep-tor is folded with the hairpin loop and dimerization interface on domain II (green) asso-ciated with domain IV (orange) Binding of EGF (red) induces a conformation change that exposes the dimerization interface and promotes formation of the activated dimer complex (pdb id 1NQL) (b) Dimerization involves dove-tailing of the domain II

hair-pin loops (pdb id  1IVO).

Phosphorylated tyrosines

Downstream signaling molecules

Ligand (e.g., EGF)

Ligand-induced dimerization

Y992

Y1173 Y1148 Y1068 Y1045

1.

Activation of kinase domain

Extracellular

region (1–621)

Kinase domain

(686–960)

Sites of tyrosine

phosphorylation

Juxtamembrane

segment (645–685)

Transmembrane

segment (622–644)

2.

FIGURE 32.13 Hormone-induced dimerization of the EGF receptor promotes autophosphorylation

of five tyrosine residues near the C-terminus of each EGF receptor subunit Signaling proteins that contain phosphotyrosine-binding SH2 domains can be activated by binding to these phosphotyrosines.

FIGURE 32.12 (a) The kinase domain of the EGF receptor consists of an N-terminal lobe built around a twisted

-sheet and an -helical C-terminal lobe Bound ATP is pink, and the activation loop, shown blocking the

active site, is blue (b) Hormone-induced receptor dimerization promotes formation of an asymmetric dimer of

the intracellular kinase domains (yellow and green) (c) Before hormonal activation (left), the kinase active site

is blocked by a 30-residue activation loop (green) In the activated complex (right), the activation loop (green)

has rotated away from the active site in one of the subunits, making room for binding of a peptide substrate

(blue) (a, b, c [right]: pdb id  2GS6; c [left]: pdb id  2GS7).

The Insulin Receptor Mediates Several Signaling Pathways

Insulin, a small heterodimeric peptide (see Figure 5.8), is the most potent anabolic hormone known It regulates blood glucose levels, and it promotes the synthesis and storage of carbohydrates, proteins, and lipids Abnormalities of insulin pro-duction, action, or both lead to diabetes, as well as other serious health issues In-sulin binding to receptors in liver, muscle, and other tissues triggers multiple sig-naling pathways, and insulin action is responsible for a variety of cellular effects The insulin receptor is an RTK that catalyzes the phosphorylation of tyrosine

residues of several intracellular substrates, including the insulin receptor substrate (IRS) proteins, and several other proteins known as Gab-1, Shc, and APS (Figure

32.14) Each of these phosphorylated substrates binds a particular family of proteins containing SH2 domains (see Figures 32.5 and 32.6) These SH2 proteins interact specifically with phosphotyrosine-containing IRS sequences Each of these signaling pathways can be confined to distinct cellular locations and can proceed with a dif-ferent time course, thus providing spatial and temporal dimensions to insulin ac-tion in cells

The Insulin Receptor Adopts a Folded Dimeric Structure

in the Membrane

Unlike the majority of RTKs, which are single-chain receptors, the insulin receptor

is an 22tetramer The -chain contains two leucine-rich domains (L1 and L2)

with a cysteine-rich domain (CR) between them, as well as an intact fibronectin do-main (Fn1) and a partial fibronectin dodo-main (Fn2) The -chain contains the other

half of Fn2, followed by a third fibronectin domain (Fn3), a transmembrane

-helix, and (inside the cell) the tyrosine kinase domain (see Figure 32.9) The

ex-tracellular domain (the ectodomain) forms a folded dimer, with the L1 and L2

domains of one -chain juxtaposed with the Fn2 domain of the other / chain pair, to create the binding site (Figure 32.15) Thus, each of the two insulin-binding sites of the ectodomain consists of portions of both -chains.

Autophosphorylation of the Insulin Receptor Kinase Opens the Active Site

Binding of insulin to its receptor activates the tyrosine kinase activity of the intra-cellular domains Like the EGF receptor kinase, the insulin receptor kinase con-tains an activation loop that lies across the kinase active site, excluding substrate peptides and thus inhibiting the kinase Phosphorylation of three tyrosine residues

CAP

Grb2

Insulin receptor

Shc

Shp-2

MAP kinase pathway

Gab-1 Y-P

P-Y

P13-K IRS1-4

CrkII

Lipid raft

AKT pathway

Cbl

P-Y APS

FIGURE 32.14 Substrates of the insulin receptor tyrosine

kinase include the insulin receptor substrate (IRS)

pro-teins, as well as Gab-1, Shc, and APS The phosphorylated

substrates in turn bind to several families of SH2

domain–containing proteins, activating several

signal-ing pathways.

Insulin-binding

sites

L1

L2

CR

Fn1

Fn2

Fn3

FIGURE 32.15 The ectodomain formed by the insulin

receptor is constructed from the two / units These two

units are folded so that the L1 domain of one -chain is

juxtaposed with the Fn1 domain of the other -chain.

Each insulin-binding site is created by the L1 and L2

domains of one -chain and the Fn2 domain of the

other/ chain pair (see Figure 32.9) (pdb id  2DTG).

on the activation loop—another case of autophosphorylation—causes the

activa-tion loop to move out of and away from the active site (Figure 32.16) This opens

the active site so that target proteins are bound and phosphorylated by the kinase,

triggering the appropriate signaling pathways (see Figure 32.14)

Receptor Guanylyl Cyclases Mediate Effects of Natriuretic Hormones

When you eat a salty meal, your body secretes hormones that protect you from the

harmful effects of excess salt intake When your heart senses that blood volume is too

great, it sends signals to the kidneys to excrete NaCl and water (processes termed

natriuresis and diuresis, respectively) These are just two examples of the action of

natriuretic hormones,which allow tissues and organs to communicate with one

an-other to regulate the volumes of blood and an-other body fluids and the osmotic effects

of Na, K, Cl, and other ions Guanylin and uroguanylin are produced in the

in-testines after ingestion of a salty meal and are secreted into the intestinal lumen

Binding of guanylin and uroguanylin to receptor guanylyl cyclases on cell

mem-branes lining the lumen activates the intracellular guanylyl cyclase (Figure 32.17a)

cGMP (see Figure 10.12) produced in this reaction is a second messenger that

in-hibits Nauptake from the lumen and activates Clexport into the lumen The

re-sult is a beneficial enhanced excretion of NaCl and water (This effect can be carried

too far, however Heat-stable enterotoxin (ST) produced by E coli—with a sequence

similar to guanylin and uroguanylin—causes violent diarrhea.) Atrial natriuretic

peptide (ANP) and brain natriuretic peptide (BNP, so named because it was

discov-ered first in brain) are both produced primarily in the heart When the heart

mus-cle is stressed and stretched by increased blood volume, the heart secretes ANP and

BNP into the blood In the kidneys, ANP and BNP bind to RGCs in kidney tubules,

activating intracellular guanylyl cyclase and producing cGMP (Figure 32.17b) As in

the intestines, cGMP inhibits Nauptake, once again stimulating excretion of salt

and water and reducing blood volume

A Symmetric Dimer Binds an Asymmetric Peptide Ligand

RGC monomers associate as dimers in the membrane, even in the absence of their

hormone ligands (Figure 32.18a) A dimeric receptor complex is activated by the

binding of a single polypeptide hormone This raises two important questions about

the mechanism of action of these receptors: (1) How does an asymmetric hormone

ligand (for example, ANP) bind to a symmetric homodimeric receptor? And (2) how

does hormone binding to its extracellular dimeric receptor activate the intracellular

guanylyl cyclase domain? Answers to these provocative questions have been provided

FIGURE 32.16In its inactive state, the insulin receptor tyrosine kinase is inhibited by an activation loop (yellow and red), which prevents substrate access to the active site (left—pdb id  1IRK) Autophosphorylation of three tyrosine residues on the activation loop induces a con-formation change that rotates the loop out of the active site (right—pdb id  1IR3), permitting access by insulin receptor substrates (blue) and ATP (ATP analog in orange).

by Kunio Misono and his colleagues, who determined the structure of the ANP receptor–ANP complex (Figure 32.18b) Remarkably, there is no significant in-tramolecular conformational change in either of the ANP receptor monomers How-ever, upon ANP binding, the two ANP receptor molecules undergo a twist motion in the membrane in order to insert the ANP hormone between them (Figure 32.19) The hormone lies like a disc between the two receptor subunits The hormone-induced twist of the extracellular domains suggests a mechanism for activation of guanylyl cyclase activity across the membrane Rotation of the transmembrane

+

E coli ST N S S N Y C C E L C C N P A T G C C Y Human guanylin P G T C E I C A Y A A T G C C Human uroguanylin

Intestinal cell (enterocyte)

Kidney tubule cell or

Kidney cells

Intestinal cells

N D C E C V N V A TG C C L

L D

G R I

I G S

S R L S

N S F R Y

A

S E

C Atrial

natriuretic

G

L G

Cl–

Na +

H +

cGMP

Blood Lumen

GTP

FIGURE 32.17 (a) Ingestion of a salty meal triggers excretion of NaCl and water in the intestines Binding of

guanylin and uroguanylin to RGC on cell membranes lining the lumen activates the intracellular guanylyl cyclase cGMP produced by the cyclase inhibits Nauptake from the lumen and activates Clexport into the

lumen (b) Atrial natriuretic peptide (ANP) protects the heart and circulatory system from the deleterious

effects of increased blood volume When the heart muscle is stressed and stretched, the heart secretes ANP ANP binding to RGCs in kidney tubules activates intracellular guanylyl cyclase, producing cGMP Inhibition of

Nauptake in the kidneys stimulates excretion of salt and water, reducing blood volume.

N-terminus

C-terminus

C-terminus

ANP

FIGURE 32.18 Activation of the ANP receptor involves binding of an asymmetric ligand at the interface of two

identical receptor domains Comparison of the structures of the receptor domain in the absence (a) and presence (b) of ANP reveals no significant intramolecular conformational change in either of the receptor

monomers ANP binding induces a twist of the two ANP receptor molecules, allowing the ANP hormone to insert between them (a) PDB file provided by Kunio Misono, University of Nevada, Reno; (b) pdb id  1T34.

(a)

(b)