Voet D., Voet Ju.G. Biochemistry

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Steroids, being water insoluble, are transported in the

blood in complex with the glycoprotein transcortin and, to

a lesser extent, by albumin. The steroids (including vitamin

D) spontaneously pass through the membranes of their

target cells to the cytosol, where they bind to their cognate

receptors. The steroid–receptor complexes then migrate to

the cell nucleus, where they function as transcription fac-

tors to induce, or in some cases repress, the transcription of

specific genes (a process that is discussed in Section 34-3Bn).

In this way, the glucocorticoids and the mineralocorticoids

influence the expression of numerous metabolic enzymes

in their respective target tissues. Thyroid hormones, which

are also nonpolar, function similarly. However, as we shall

see in the following sections, all other hormones act less

directly in that they bind to their cognate cell-surface re-

ceptors and thereby trigger complex cascades of events

within cells that ultimately influence transcription as well

as other cellular processes.

Impaired adrenocortical function, either through disease

or trauma,results in a condition known as Addison’s disease,

which is characterized by hypoglycemia, muscle weakness,



loss, K



retention, impaired cardiac function, loss of ap-

petite, and a greatly increased susceptibility to stress. The

victim, unless treated by the administration of glucocorti-

coids and mineralocorticoids, slowly languishes and dies

without any particular pain or distress. The opposite prob-

lem, adrenocortical hyperfunction, which is usually caused

by a tumor of the adrenal cortex or the pituitary gland (Sec-

tion 19-1H), results in Cushing’s syndrome, which is charac-

terized by fatigue, hyperglycemia, edema (water retention),

and a redistribution of body fat to yield a characteristic

“moon face.” Long-term treatments of various diseases with

synthetic glucocorticoids result in similar symptoms.

Gonadal Steroids Mediate Sexual

Development and Function

The gonads (testes in males, ovaries in females), in addi-

tion to producing sperm or ova, secrete steroid hormones

(androgens and estrogens) that regulate sexual differentiation,

Corticosterone

Aldosterone

the expression of secondary sex characteristics, and sexual

behavior patterns. Although testes and ovaries both synthe-

size androgens and estrogens, the testes predominantly se-

crete androgens, which are therefore known as male sex

hormones, whereas ovaries produce mostly estrogens,

which are consequently termed female sex hormones.

Androgens, of which testosterone is prototypic,

Section 19-1. Hormones 681

Testosterone

-Estradiol

Progesterone

lack the C

substituent at C17 present in glucocorticoids

and are therefore C

compounds. Estrogens, such as

-estradiol, resemble androgens but lack a C10 methyl group

because they have an aromatic A ring and are therefore

compounds. Interestingly, testosterone is an intermedi-

ate in estrogen biosynthesis (Section 25-6C). A second

class of ovarian steroids, C

compounds called progestins,

help mediate the menstrual cycle and pregnancy (Section

19-1I). Progesterone, the most abundant progestin, is, in

fact, a precursor of glucocorticoids, mineralocorticoids, and

testosterone (Section 25-6C).

c. Sexual Differentiation Is Both Hormonally

and Genetically Controlled

What factors control sexual differentiation? If the go-

nads of an embryonic male mammal are surgically re-

moved, that individual will become a phenotypic female.

Evidently, mammals are programmed to develop as females

unless embryonically subjected to the influence of testicular

hormones. Indeed, genetic males with absent or nonfunc-

tional cytosolic androgen receptors are phenotypic fe-

males, a condition named testicular feminization. Curi-

ously, estrogens appear to play no part in embryonic

female sexual development, although they are essential for

female sexual maturation and function.

JWCL281_c19_671-743.qxd 3/16/10 7:16 PM Page 681

Normal individuals have either the XY (male) or the

XX (female) genotypes (Section 1-4C). However, those

with the abnormal genotypes XXY (Klinefelter’s syn-

drome) and X0 (only one sex chromosome; Turner’s syn-

drome) are, respectively, phenotypic males and phenotypic

females, although both are sterile.Apparently, the normal Y

chromosome confers the male phenotype, whereas its ab-

sence results in the female phenotype. There are, however,

rare (1 in 20,000) XX males and XY females. These XX

males (who are sterile and have therefore been identified

through infertility clinics) have a small segment of a nor-

mal Y chromosome translocated onto one of their X chro-

mosomes, whereas XY females are missing this segment.

Early male and female embryos—through the sixth

week of development in humans—have identical undiffer-

entiated genitalia. Evidently, the Y chromosome contains a

gene, testes-determining factor (TDF), that induces the

differentiation of testes, whose hormonal secretions, in

turn, promote male development. The misplaced chromo-

somal segments in XY females and XX males have a com-

mon 140-kb sequence that contains a structural gene

dubbed SRY (for sex-determining region of Y) that en-

codes an 80-residue DNA-binding motif. Several sex-

reversed XY women have a mutation in the region of their

SRY gene encoding this DNA-binding domain that elimi-

nates its ability to bind DNA, a mutation that is not present

in their father’s gene. SRY is expressed in embryonic go-

nadal cells previously shown to be responsible for testis de-

termination. Moreover, of eleven XX mice that were made

transgenic for Sry (the mouse analog of SRY), three were

males. Thus, TDF/SRY is the first clear example of a mam-

malian gene that controls the development of an entire or-

gan system (development is discussed in Section 34-4B).

H. Control of Endocrine Function: The

Hypothalamus and Pituitary Gland

The anterior lobe of the pituitary gland (the adenohypo-

physis) and the hypothalamus, a nearby portion of the brain,

constitute a functional unit that hormonally controls much

of the endocrine system. The neurons of the hypothalamus

synthesize a series of polypeptide hormones known as re-

leasing factors and release-inhibiting factors which, on de-

livery to the adenohypophysis via a direct circulatory con-

nection (their half-lives are on the order of a few minutes),

stimulate or inhibit the release of the corresponding trophic

hormones into the bloodstream. Trophic hormones, by defi-

nition, stimulate their target endocrine tissues to secrete

the hormones they synthesize. Since releasing and release-

inhibiting factors, trophic hormones, and endocrine hor-

mones are largely secreted in nanogram, microgram, and

milligram quantities per day, respectively, and tend to have

progressively longer half-lives, these hormonal systems can

be said to form amplifying cascades. Four such systems are

prominent in humans (Fig. 19-8; left):

1. Corticotropin-releasing factor (CRF; 41 residues)

causes the adenohypophysis to release adrenocorticotropic

682 Chapter 19. Signal Transduction

Figure 19-8 Hormonal control circuits, indicating the

relationships between the hypothalamus, pituitary, and target

tissues. Releasing factors and release-inhibiting factors secreted

by the hypothalamus signal the adenohypophysis to secrete or

stop secreting the corresponding trophic hormones, which, for

the most part, stimulate the corresponding endocrine gland(s) to

Hypothalamus

Higher brain centers

Adenohypophysis (anterior pituitary)

CRF TRF GnRF

GRF

Somatostatin (GRIF)

ACTH TSH FSH Growth hormone

Adrenocortical

hormones

& T

Androgens SomatomedinsEstrogens,

progestins

Adrenal cortex Thyroid Testes/Ovaries Liver

Muscles, liver, and other tissues + sex accessory tissues + bones

Central nervous system

Neurohypophysis (posterior pituitary)

Water resorption Uterine contraction

Vasopressin Oxytocin

Feedback

inhibition

Feedback

inhibition

secrete their respective endocrine hormones.The endocrine

hormones, in addition to controlling the growth, differentiation,

and metabolism of their corresponding target tissues, influence

the secretion of releasing factors and trophic hormones through

feedback inhibition.The levels of trophic hormones likewise

influence the levels of their corresponding releasing factors.

JWCL281_c19_671-743.qxd 3/16/10 7:16 PM Page 682

hormone (ACTH; 39 residues), which stimulates the re-

lease of adrenocortical steroids. The entire system is under

feedback control:ACTH inhibits the release of CRF and the

adrenocortical steroids inhibit the release of both CRF and

ACTH. Moreover, the hypothalamus, being part of the

brain, is also subject to neuronal control, so the hypothala-

mus forms the interface between the nervous system and the

endocrine system.

2. Thyrotropin-releasing factor (TRF), a tripeptide

with an N-terminal pyroGlu residue (a Glu derivative in

which the side chain carboxyl group forms an amide bond

with its amino group),

stimulates the adenohypophysis to release the trophic hor-

mone thyrotropin (thyroid-stimulating hormone; TSH)

which, in turn, stimulates the thyroid to synthesize and re-

lease T

and T

. TRF, as are other releasing factors, is pres-

ent in the hypothalamus in only vanishingly small quanti-

ties. It was independently characterized in 1969 by Roger

Guillemin and Andrew Schally using extracts of the hypo-

thalami from over 2 million sheep and 1 million pigs.

3. Gonadotropin-releasing factor (GnRF; 10 residues)

stimulates the adenohypophysis to release luteinizing hor-

mone (LH) and follicle-stimulating hormone (FSH), which

are collectively known as gonadotropins. In males, LH

stimulates the testes to secrete androgens, whereas FSH

promotes spermatogenesis. In females, FSH stimulates the

development of ovarian follicles (which contain the imma-

ture ova), whereas LH triggers ovulation.

4. Growth hormone-releasing factor (GRF; 44 residues)

and somatostatin [14 residues; also known as growth hor-

mone release-inhibiting factor (GRIF)], stimulate/inhibit

the release of growth hormone (GH) from the adenohy-

pophysis. GH (also called somatotropin), in turn, stimu-

lates generalized growth (see Fig. 5-5 for a striking example

of its effect). GH directly accelerates the growth of a vari-

ety of tissues (in contrast to TSH, LH, and FSH, which act

only indirectly by activating endocrine glands) and induces

the liver to synthesize a series of polypeptide growth fac-

tors termed somatomedins that stimulate cartilage growth

and have insulinlike activities.

TSH, LH, and FSH are heterodimeric glycoproteins, which

in a given species, all have the same  subunit (92 residues)

and a homologous  subunit (114, 114, and 118 residues,

respectively, in humans). Human GH consists of a single

191-residue polypeptide chain, which is unrelated to TSH,

LH, or FSH.

Gonadotropin-releasing factor (GnRF)

pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH

110

CH C

His NH

Pro

pyroGlu

Thyrotropin-releasing factor (TRF)

a. The Neurohypophysis Secretes Oxytocin

and Vasopressin

The posterior lobe of the pituitary, the neurohypophy-

sis, which is anatomically distinct from the adenohypophy-

sis, secretes two homologous nonapeptide hormones (Fig.

19-8, right): vasopressin [also known as antidiuretic hor-

mone (ADH)], which increases blood pressure and stimu-

lates the kidneys to retain water; and oxytocin, which

causes contraction of uterine smooth muscle and therefore

induces labor:

The rate of vasopressin release is largely controlled by os-

moreceptors, which monitor the osmotic pressure of the

blood.

I. Control of the Menstrual Cycle

The menstrual cycle and pregnancy are particularly illus-

trative of the interactions among hormonal systems. The

⬃28-day human menstrual cycle (Fig. 19-9) begins during

menstruation with a slight increase in the FSH level that

initiates the development of a new ovarian follicle. As the

follicle matures, it secretes estrogens that act to sensitize

the adenohypophysis to GnRF. This process culminates in

a surge of LH and FSH, which triggers ovulation. The rup-

tured ovarian follicle, the corpus luteum, secretes proges-

terone and estrogens, which inhibit further gonadotropin

Human vasopressin

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH

Human oxytocin

Cys-Tyr- Ile - Gln-Asn-Cys-Pro-Leu-Gly-NH

Section 19-1. Hormones 683

Figure 19-9 Patterns of hormone secretion during the

menstrual cycle in the human female.

2 6 10 14128418222016 24 26 28

Hormone level

Menstrual cycle (days)

OvulationEstradiol

FSH

Progesterone

LH surge

JWCL281_c19_671-743.qxd 3/16/10 7:16 PM Page 683

secretion by the adenohypophysis and stimulate the uter-

ine lining to prepare for the implantation of a fertilized

ovum. If fertilization does not occur, the corpus luteum re-

gresses, progesterone and estrogen levels fall, and menstru-

ation (the sloughing off of the uterine lining) ensues. The

reduced steroid levels also permit a slight increase in the

FSH level, which initiates a new menstrual cycle.

A fertilized ovum that has implanted into the hormon-

ally prepared uterine lining soon commences synthesizing

chorionic gonadotropin (CG). This heterodimeric glyco-

protein hormone contains a 145-residue ␤ subunit that has

a high degree of sequence identity with those of LH (85%),

FSH (45%), and TSH (36%) over their N-terminal 114

residues and the same ␣ subunit. CG stimulates the corpus

luteum to continue secreting progesterone rather than re-

gressing and thus prevents menstruation. Pregnancy tests

utilize immunoassays that can detect CG in blood or urine

within a few days after embryo implantation. Most female

oral contraceptives (birth control pills) contain proges-

terone derivatives, whose ingestion induces a state of

pseudopregnancy in that they inhibit the midcycle surge of

FSH and LH so as to prevent ovulation.

J. Growth Hormone and Its Receptor

The binding of growth hormone activates its receptor to

stimulate growth and metabolism in muscle, bone, and car-

tilage cells.This 620-residue receptor is a member of a large

family of structurally related protein growth factor recep-

tors, which includes those for various interleukins (Section

19-3Eb).All of these receptors consist of an N-terminal ex-

tracellular ligand-binding domain, a single transmembrane

segment that is almost certainly helical, and a C-terminal

cytoplasmic domain that is not homologous within the su-

perfamily but in many cases contains a tyrosine kinase func-

tion (Section 19-3A).

The X-ray structure of the 191-residue human growth hor-

mone (hGH) in complex with the 238-residue ectodomain

(extracellular portion; Greek: ectos, outside) of its binding

protein (hGHbp), determined by Abraham de Vos and

Anthony Kossiakoff, revealed that this complex consists of

two molecules of hGHbp bound to a single hGH molecule

(Fig. 19-10). hGH consists largely of an up–up–down–down

four-helix bundle, which closely resembles that in the

previously determined X-ray structure of porcine GH,

although with significant differences that may be caused by

the binding of hGH to its receptor.A variety of other pro-

tein growth factors with known structures, including many

interleukins (Section 19-3Eb), contain similar four-helix

bundles. Each hGHbp molecule consists of two structurally

similar ⬃100-residue fibronectin type III domains, each of

which forms a topologically identical sandwich of a three-

and a four-stranded antiparallel ␤ sheet that resembles the

immunoglobulin fold (Fig. 8-48). Fibronectin type III do-

mains, so called because they were first observed in the

multidomain extracellular matrix glycoprotein fibronectin,

are among the most common structural modules in recep-

tor ectodomains.

The two hGHbp molecules bind to hGH with near 2-fold

symmetry about an axis that is roughly perpendicular to

the helical axes of the hGH four-helix bundle and, pre-

sumably, to the plane of the cell membrane to which the in-

tact hGH receptor is anchored (Fig. 19-10).The C-terminal

domains of the two hGHbp molecules are almost parallel

and in contact with one another. Intriguingly, the two

hGHbp molecules use essentially the same residues to bind

to sites that are on opposite sides of hGH’s four-helix bun-

dle and that have no structural similarity. The X-ray struc-

ture is largely consistent with the results of mutational

studies designed to identify the hGH and hGHbp residues

important for receptor binding.

The ligand-induced dimerization of hGHbp has im-

portant implications for the mechanism of signal trans-

duction. The dimerization, which does not occur in the

absence of hGH, apparently brings together the intact re-

ceptors’ intracellular domains in a way that activates an

effector protein such as a tyrosine kinase (Section 19-3A).

Indeed, hGH mutants that cannot induce receptor

684 Chapter 19. Signal Transduction

Figure 19-10 X-ray structure of human growth hormone

(hGH) in complex with two molecules of its receptor’s

extracellular domain (hGHbp). The proteins are drawn in ribbon

form, with the hGH magenta and the two hGHbp molecules,

which together bind one molecule of hGH, each colored in

rainbow order from N-terminus (blue) to C-terminus (red).The

view is along the axis of the hGH’s four-helix bundle with the

approximate 2-fold axis relating the two hGHbp molecules

vertical.The dotted red lines represent the pathways that the

hGHbp chains take in penetrating the membrane. Note that the

width of a membrane is actually nearly the ⬃75-Å height of the

hGHbp–hGH–hGHbp complex. [Based on an X-ray structure by

Abraham de Vos and Anthony Kossiakoff, Genentech Inc., South

San Francisco, California. PDBid 3HHR.]

See Interactive

Exercise 11

JWCL281_c19_671-743.qxd 10/19/10 7:35 AM Page 684

dimerization are biologically inactive. Numerous other

protein growth factors also induce the dimerization of

their receptors.

a. Abnormal GH Production Causes

Abnormal Growth

Overproduction of GH, usually a consequence of a pi-

tuitary tumor, results in excessive growth. If this condition

commences while the skeleton is still growing, that is, be-

fore its growth plates have ossified, then this excessive

growth is of normal proportions over the entire body, re-

sulting in gigantism. Moreover, since excessive GH in-

hibits the testosterone production necessary for growth

plate ossification, such “giants” continue growing through-

out their abnormally short lives. If, however, the skeleton

has already matured, GH stimulates only the growth of

soft tissues, resulting in enlarged hands and feet and thick-

ened facial features, a condition named acromegaly (Fig.

19-11). The opposite problem, GH deficiency, which re-

sults in insufficient growth (dwarfism), can be treated be-

fore skeletal maturity by regular injections of hGH (ani-

mal GH is ineffective in humans). hGH was, at first,

available only from the pituitaries of cadavers and there-

fore was in extremely short supply. Since the early 1980s,

however, hGH has been synthesized in virtually unlimited

amounts via recombinant DNA techniques. Indeed, hGH

has been illicitly taken by individuals wishing to increase

their athletic prowess (the ban on using hGH could not be

enforced until the early 2000s, when blood tests were

developed that could distinguish between natural and

recombinant hGH).

K. Opioid Peptides

Among the most intriguing hormones secreted by the

adenohypophysis are polypeptides that have opiatelike ef-

fects on the central nervous system. These include the

31-residue ␤-endorphin, its N-terminal pentapeptide, termed

methionine-enkephalin, and the closely similar leucine-

enkephalin (although the enkaphalins are independently

expressed).

These substances bind to opiate receptors in the brain and

have been shown to be their physiological agonists. The

role of these so-called opioid peptides has yet to be defini-

tively established, but it appears they are important in the

control of pain and emotional states. Pain relief through

the use of acupuncture and placebos as well as such phe-

nomena as “runner’s high” may be mediated by opioid

peptides.

L. The Hormonal Function of Nitric Oxide

Nitric oxide (NO) is a reactive and toxic free radical gas.

Thus, it came as a great surprise that this molecule functions

as paracrine signal in regulating blood vessel dilation and

serves as a neurotransmitter. It also functions in the immune

response. The role of NO in vasodilation was discovered

Tyr - Gly - Gly-Phe-Met-Thr - Ser - Glu - Lys-Ser

Gln-Thr-Pro -Leu-Val - Thr -Leu-Phe-Lys-Asn

Ala - Ile - Val-Lys -Asn -Ala -His -Lys - Lys -Gly

Gln-

Tyr - Gly - Gly-Phe-Leu

11 15

␤-Endorphin

Leucine-enkephalin (Leu-enkephalin)

Tyr - Gly - Gly-Phe-Met

Methionine-enkephalin (Met-enkephalin)

Morphine (an opiate)

Section 19-1. Hormones 685

Figure 19-11 Acromegaly. The characteristic enlarged features

of Akhenaten, the Pharaoh who ruled Egypt in the years

1379–1362

B.C., strongly suggest that he suffered from

acromegaly. [Bildarchiv Preussischer Kulturbesitz/Art Resource.]

JWCL281_c19_671-743.qxd 6/30/10 1:48 PM Page 685

through the observation that substances such as acetyl-

choline (Section 20-5Cb) and bradykinin (Section 7-5B),

which act through the phosphoinositide signaling system

(Section 19-4) to increase the flow through blood vessels

by eliciting smooth muscle relaxation, require an intact en-

dothelium overlying the smooth muscle (the endothelium is

a layer of cells that lines the inside of certain body cavities

such as blood vessels). Evidently, endothelial cells respond

to the presence of these vasodilation agents by releasing

a diffusible and highly labile substance (half-life ⬃5 s) that

induces the relaxation of smooth muscle cells. This sub-

stance was identified as NO, in part, through parallel stud-

ies identifying NO as the active metabolite that mediates

the well-known vasodilating effects of antianginal organic

nitrates such as nitroglycerin

(angina pectoris is a condition caused by insufficient blood

flow to the heart muscle, leading to severe chest pain; nitro-

glycerin’s vasodilating properties were discovered in the

nineteenth century through the observation that workers

with angina pectoris in a factory that produced nitroglycerin

experienced greater pain on weekends).

a. Nitric Oxide Synthase Requires Five

Redox-Active Cofactors

NO is synthesized by NO synthase (NOS), which cat-

alyzes the NADPH-dependent 5-electron oxidation of

L-arginine by O

to yield NO and the amino acid L-citrulline

with the intermediate formation of N



-hydroxy-L-arginine

(NOHA; Fig. 19-12). Three isozymes of NOS have been

identified in mammals, neuronal NOS (nNOS), in-

ducible NOS (iNOS), and endothelial NOS (eNOS), which

are also known as NOS-1, -2, and -3, respectively. These

isozymes, which have 50 to 60% sequence identity, are all

Nitroglycerin

homodimeric proteins of 125- to 160-kD subunits that each

consist of two domains:

1. An N-terminal, ⬃500-residue, oxygenase or heme

domain that catalyzes both reaction steps of Fig. 19-12

and contains the dimer interface. This domain binds the

substrates O

and L-arginine and two redox-active pros-

thetic groups, Fe(III)-heme and 5,6,7,8-tetrahydrobiopterin

B),

a compound that also functions in the hydroxylation of

phenylalanine to tyrosine (Section 26-3Ha). The X-ray

structures of the oxygenase domains of nNOS (Fig. 19-13a),

iNOS, and eNOS are closely similar.

2. A C-terminal, ⬃600-residue, reductase domain that

supplies the electrons for the NOS reaction. It binds

NADPH and two redox-active prosthetic groups, an FAD

(Fig. 16-8) and a flavin mononucleotide (FMN; FAD lack-

ing its AMP residue; Fig. 22-17a) via three nucleotide bind-

ing modules. This domain is homologous to cytochrome

P450 reductase, an enzyme that participates in detoxifica-

tion processes (Section 15-4Bc).The X-ray structure of the

reductase domain of nNOS, determined by John Tainer and

Elizabeth Getzoff, is shown in Fig. 19-13b.

NADPH bound to the reductase domain transmits its

electrons via the FAD and then the FMN to the heme in

the oxygenase domain. Interestingly, the reductase do-

main transmits its electrons to the oxygenase domain on

the opposite subunit. This was shown by Dennis Stuehr

through his construction of an NOS heterodimer in which

one subunit was full length and the other consisted of only a

oxygenase domain. If a mutation that disrupts the

L-arginine

5,6,7,8-Tetrahydrobiopterin

686 Chapter 19. Signal Transduction

Figure 19-12 The NO synthase (NOS) reaction. The N



-hydroxy-L-arginine intermediate is

tightly bound to the enzyme.



COO





-Arginine



COO







-Hydroxy-

-arginine (NOHA)



COO



-Citrulline

NADPH

 O

NADP



 H

NADPH

 O

NADP



 H

JWCL281_c19_671-743.qxd 3/16/10 7:16 PM Page 686

binding site was in the full-length subunit, the enzymatic

activity of this heterodimer was unaffected, but if it

was in the oxygenase domain–only subunit, activity was

abolished.

The heme Fe atom is 5-coordinated with its axial ligand

supplied by a specific Cys S atom.The

L-arginine substrate

binds on the opposite side of the heme from this Cys with

the N atom to be hydroxylated ⬃4.0 Å distant from the Fe

atom, a distance too large for covalent bond formation.

Since O

is known to react with the heme Fe atom, it pre-

sumably binds between it and this N atom.

NOS requires bound H

B to produce NO. In the ab-

sence of this prosthetic group, NOS efficiently catalyzes the

NADPH-mediated oxidation of O

to H

. Investigations

by Steuhr have established that H

B functions as an inter-

nal redox agent in that, during the reactions forming both

NOHA and NO, it is oxidized to its radical form (H

ⴢ

⫹

)

and then re-reduced to its reduced form (H

B). Thus, H

does not undergo net oxidation in the NOS reaction (as it

does in the reaction hydroxylating phenylalanine to tyro-

sine; Section 26-3Ha).

NO rapidly diffuses across cell membranes, although its

high reactivity prevents it from traveling ⬎1 mm from its

site of synthesis (in particular, it efficiently reacts with both

oxyhemoglobin and deoxyhemoglobin: NO ⫹ HbO

⫺

⫹ metHb; and NO ⫹ Hb S HbNO; Section 10-1A).

The physiological target of NO in smooth muscle cells is

guanylate cyclase (GC), which catalyzes the reaction of

GTP to yield 3ⴕ,5ⴕ-cyclic GMP (cGMP),

an intracellular second messenger that resembles 3¿,5¿-cyclic

AMP (cAMP; GC is a homolog of adenylate cyclase;

Section 19-2D). cGMP causes smooth muscle relaxation

through its stimulation of protein phosphorylation by

cGMP-dependent protein kinase. NO reacts with GC’s

heme prosthetic group to yield nitrosoheme, whose pres-

ence increases GC’s activity by up to 200-fold, presumably

via a conformation change resembling that in hemoglobin

on binding O

(Section 10-2Ba; although GC binds O

quite poorly).

3⬘,5ⴕ-Cyclic GMP

(cGMP)

⫺

O OH

Section 19-1. Hormones 687

(a)

(b)

Figure 19-13 X-ray structure of rat nNOS. (a) Its N-terminal

oxygenase domain in complex with heme, L-arginine, and H

(b) Its C-terminal reductase domain in complex with FMN, FAD,

and NADP

⫹

. In both structures the homodimeric protein is

viewed in semitransparent ribbon form with its 2-fold axis

vertical and with one subunit colored in rainbow order from

N-terminus (blue) to C-terminus (red) and the other subunit

blue-gray. The various bound groups are drawn in space-filling

form colored according to atom type (heme and FMN C green,

L-arginine and FAD C cyan, H

B and NADP

⫹

C magenta, N

blue, O red, P orange, and Fe red-brown). [Part a based on an

X-ray structure by Thomas Poulos, University of California at

Irvine; and Part b based on an X-ray structure by John Tainer

and Elizabeth Getzoff, The Scripps Research Institute, La Jolla,

California. PDBids 1OM4 and 1TLL.]

JWCL281_c19_671-743.qxd 6/4/10 10:52 AM Page 687

b. eNOS and nNOS but Not iNOS Are

Regulated by [Ca

2ⴙ

]

2⫹

–calmodulin activates eNOS and nNOS by binding

to the ⬃30-residue segments linking their oxygenase and

reductase domains. Thus, for example, the stimulatory ac-

tion of vasodilatory agents on the phosphoinositide signal-

ing system (Section 19-4A) in endothelial cells to produce

an influx of Ca

2⫹

results in the synthesis of NO. Hence, NO

functions to transduce hormonally induced increases in in-

tracellular [Ca

2⫹

] in endothelial cells to increased rates of

production of cGMP in neighboring smooth muscle cells.

NO produced by nNOS mediates vasodilation through

endothelium-independent neural stimulation of smooth

muscle. In this signal transduction pathway, which is re-

sponsible for the dilation of cerebral and other arteries as

well as penile erection (see Section 19-2E), nerve impulses

cause an increased [Ca

2⫹

] in nerve terminals, thereby stim-

ulating neuronal NOS. The resultant NO diffuses to nearby

smooth muscle cells, where it binds to guanylate cyclase

and activates it to synthesize cGMP as described above.

Inducible NOS (iNOS) is unresponsive to Ca

2⫹

even

though it has two tightly bound calmodulin subunits. How-

ever, it is transcriptionally induced in macrophages and

neutrophils (white blood cells that function to ingest and

kill bacteria), as well as in endothelial and smooth muscle

cells (in contrast, eNOS and nNOS are expressed constitu-

tively, that is, at a constant rate). Several hours after expo-

sure to cytokines (protein growth factors that regulate the

differentiation, proliferation, and activities of many types

of cells; Section 19-3Eb) and/or endotoxins (bacterial cell

wall lipopolysaccharides that elicit inflammatory re-

sponses; Section 35-2Fb), these cells begin to produce large

quantities of NO and continue to do so for many hours.Ac-

tivated macrophages and neutrophils also produce super-

oxide ion (O

⫺

), which chemically combines with NO to

form the even more toxic peroxynitrite (OONO

⫺

, which

rapidly reacts with H

O to yield the highly reactive hydrox-

ide radical, OHⴢ, and NO

) that they use to kill ingested

bacteria. Indeed, NOS inhibitors block the cytotoxic ac-

tions of macrophages.

Cytokines and endotoxins induce a long-lasting and

profound vasodilation and a poor response to vasocon-

strictors such as epinephrine. The sustained release of NO

has been implicated in septic shock (an often fatal immune

system overreaction to bacterial infection that results in a

catastrophic reduction in blood pressure), in inflamma-

tion-related tissue damage as occurs in autoimmune dis-

eases such as rheumatoid arthritis, and in the damage to

neurons in the vicinity of but not directly killed by a stroke

(reperfusion injury; Section 10-1Aa). Many of these condi-

tions might be alleviated if drugs can be developed that se-

lectively inhibit iNOS and/or nNOS, while permitting

eNOS to carry out its essential function of maintaining

vascular tone. Moreover, the administration of NO itself

appears to be medically useful. For example, the inhala-

tion of low levels NO has been used to reduce pulmonary

hypertension (high blood pressure in the lung, an often fa-

tal condition caused by constriction of its arteries) in new-

born infants.

2 HETEROTRIMERIC G PROTEINS

We have seen (Section 18-3) that hormones such as

glucagon and epinephrine regulate glycogen metabolism

by stimulating adenylate cyclase (AC) to synthesize the

second messenger cAMP from ATP. The cAMP then

binds to protein kinase A (PKA) so as to activate this en-

zyme to initiate cascades of phosphorylation/dephospho-

rylation events that ultimately control the activities of

glycogen phosphorylase and glycogen synthase. Numer-

ous other extracellular signaling molecules (known ago-

nists, ligands, or effectors) also activate the intracellular

synthesis of cAMP, thereby eliciting a cellular response.

But what is the mechanism through which the binding of

an agonist to a receptor induces AC to synthesize cAMP

in the cytosol? In answering this question we shall see

that the systems that link receptors to AC as well as other

effectors have a surprising complexity that endows them

with immense capacity for both signal amplification and

regulatory flexibility.

A. Overview

See Guided Exploration 16: Mechanisms of hormone signaling in-

volving the adenylate cyclase system Adenylate cyclase, which is

located on the plasma membrane’s cytosolic surface, and

the receptors that activate it, whose agonist-binding sites

are exposed to the extracellular space, are separate pro-

teins that do not physically interact. Rather, they are func-

tionally coupled by heterotrimeric G proteins (Fig. 19-14),

so called because they specifically bind the guanine nu-

cleotides GTP and GDP.

AC is activated by a heterotrimeric G protein (often

called just a G protein) but only when the G protein is

complexed with GTP. However, G protein slowly hy-

drolyzes GTP to GDP ⫹ P

(at the leisurely rate of 2–3

min

⫺1

) and thereby deactivates itself (if G proteins were

efficient enzymes, they would be unable to effectively acti-

vate AC). G protein is reactivated by the exchange of its

bound GDP for GTP, a process that is mediated by the

agonist–receptor complex but not by unoccupied receptor.

Heterotrimeric G protein therefore mediates the transduc-

tion of an extracellular signal to an intracellular signal (the

cAMP). Moreover, the receptor–G protein–AC system am-

plifies the extracellular signal because each agonist–receptor

complex activates many G proteins before it is inactivated

by the spontaneous dissociation of the agonist and, during

its lifetime,each G protein ⴢ GTP–AC complex catalyzes the

formation of many cAMP molecules. In this section, we dis-

cuss how this process occurs.

Heterotrimeric G proteins are members of the super-

family of regulatory GTPases that are collectively known

as G proteins (whether one is referring to a heterotrimeric

or some other species of G protein is usually clear from

context). G proteins other than heterotrimeric G proteins

have a wide variety of essential functions including signal

transduction (e.g., Ras; Section 19-3Cf), vesicle trafficking

(e.g.,Arf, dynamin, and Rab; Sections 12-4Cd and 12-4Db),

translation (as ribosomal accessory factors; Section 32-3),

and targeting [as components of the signal recognition

688 Chapter 19. Signal Transduction

JWCL281_c19_671-743.qxd 6/4/10 10:52 AM Page 688

particle (SRP) and the SRP receptor; Section 12-4Ba].The

many G proteins share common structural motifs that bind

guanine nucleotides (GDP and GTP) and catalyze the hy-

drolysis of GTP to GDP  P

(see below).

B. G Protein-Coupled Receptors

The receptors responsible for activating AC and other tar-

gets of heterotrimeric G proteins are all integral proteins

with 7 transmembrane helices (Fig. 19-15) that have their

N-termini in the extracellular space and their C-termini in

the cytosol. These G protein-coupled receptors (GPCRs;

also called heptahelical, 7TM, and serpentine receptors)

constitute one of the largest known protein families (800

species in humans, which constitutes 3.5% of the ⬃23,000

genes in the human genome). They include receptors for

nucleosides, nucleotides, Ca

2

, catecholamines [epineph-

rine and norepinephrine as well as dopamine (Section

26-4B)] and other biogenic amines (e.g., histamine and

serotonin; Section 26-4B), eicosanoids (prostaglandins,

prostacyclins, thromboxanes, leukotrienes, and lipoxins,

derivatives of the C

fatty acid arachidonic acid, which are

potent local mediators of numerous important physiologi-

cal processes; Section 25-7), and for most of the large vari-

ety of peptide and protein hormones discussed in Section

19-1. In addition, GPCRs have important sensory func-

tions: They constitute the olfactory (odorant) and gusta-

tory (taste) receptors (of which there are estimated to be

460 different types in humans), as well as the several light-

sensing proteins in the retina, which are known as

rhodopsins. In addition, the GPCRs constitute the most

important class of drug targets in the pharmaceutical arse-

nal (Section 15-4): ⬃50% of approved drugs elicit their

therapeutic effects by selectively interacting with specific

GPCRs.

a. GPCRs Have Similar Structures

Despite the foregoing, the structures of only few species

of GPCRs have yet been elucidated, mainly due to the ap-

parent flexibilities of their extramembranous segments and

the general difficulty of crystallizing transmembrane

proteins (Section 12-3Aa). The first GPCR whose X-ray

Section 19-2. Heterotrimeric G Proteins 689

Figure 19-14 Activation/deactivation cycle for hormonally

stimulated AC. (a) In the absence of hormone, heterotrimeric G

protein binds GDP and AC is catalytically inactive. (b) The

hormone–receptor complex stimulates the G protein to

exchange its bound GDP for GTP. (c) The G protein  GTP

(a)

(b)(d)

(c)

Receptor

G protein

Hormone

Adenylate

cyclase

GDP

GTP

ATP

GDP

GTP

cAMP + PP

Cellular responses

Extracellular space

Cytoplasm

1735246

Plasma

membrane

Figure 19-15 General structure of a G protein-coupled

receptor (GPCR).

complex, in turn, binds to and thereby activates AC to produce

cAMP. (d) The eventual G protein-catalyzed hydrolysis of its

bound GTP to GDP causes G protein to dissociate from and

hence deactivate AC.

JWCL281_c19_671-743.qxd 3/16/10 7:16 PM Page 689

structure was reported was bovine rhodopsin (Fig. 19-16).

Rhodopsin consists of the 348-residue protein opsin that is

covalently linked to the chromophore retinal (Fig. 12-24)

via a Schiff base to Lys 296, much as occurs in the homolo-

gous bacteriorhodopsin (Section 12-3Ab),which is a hepta-

helical light-driven proton pump (Section 22-3Bh). The

absorption of a photon causes the rhodopsin-bound retinal

to isomerize from its ground state 11-cis form to its all-trans

form. This isomerization is accompanied by a transient con-

formational change in opsin before the all-trans-retinal is

hydrolyzed and dissociated from the opsin (which is

subsequently regenerated by the addition of 11-cis-retinal

delivered from adjacent epithelial cells in the retina). It

is this conformational change, which occurs mainly on

rhodopsin’s cytosolic surface, that activates its cognate G

protein.

Rhodopsins are unique among GPCRs in that their 11-cis

retinal “agonist” is covalently bound to the protein. All

other GPCRs bind diffusible ligands. The X-ray structures

of 

- and 

-adrenergic receptors and the human A

adenosine receptor, the only other species of GPCRs

whose structures have yet been determined, reveal that

their transmembrane portions closely resemble each other

and that of rhodopsin, and that their bound ligands occupy

positions similar to that of retinal in rhodopsin.Note that the

transmembrane helices of the GPCRs are more or less uni-

form in size (20–27 residues), but their extramembranous

segments, which largely form their ligand- and G protein-

binding sites, vary widely in length with the identity of the

GPCR (7–595 residues for the N- and C-termini and 5–230

residues for the loops connecting their TM helices).

C. Heterotrimeric G Proteins: Structure

and Function

Heterotrimeric G proteins, which were first characterized

by Alfred Gilman and Martin Rodbell, are more complex

than Fig. 19-14 implies: They consist, as their name indi-

cates, of three different subunits, ,,and  (45, 37,and 9 kD,

respectively), of which it is G



that binds GDP and GTP

(Fig. 19-17) and hence is a member of the G protein

superfamily. The binding of G



 GDP–G





to its cognate

ligand–GPCR complex induces the G



to exchange its

bound GDP for GTP and, in so doing, to dissociate from





. In contrast, G



and G



bind one another with such

high affinity that they only dissociate under denaturing

conditions. Consequently, we shall henceforth refer to their

complex as G



Both G



and G



are membrane-anchored proteins: G



through its myristoylation or palmitoylation or both at or

near its N-terminus (Section 12-3Bb), and G



through the

prenylation of G



at its C-terminus (Section 12-3Ba).These

lipid modifications stabilize the interactions of G



with



, as they localize both to the inner surface of the plasma

membrane.

GTP binding, in addition to decreasing G



’s affinity for

its cognate ligand–GPCR complex, increases its affinity for

its effector,AC.Thus, it is the binding of G



 GTP that acti-

vates AC (Fig. 19-17, left).



can also directly participate in signal transduction:

It activates a wide variety of signaling proteins including

several isoforms of AC (Section 19-2D), certain Na



and Ca

2

-specific ion channels, various protein tyrosine ki-

nases (Section 19-3A), and phospholipase C- (PLC-; a

component of the phosphoinositide signaling system; Sec-

tion 19-4Ba). G



thereby provides an important source of

cross talk between signaling systems.

On the eventual G



-catalyzed hydrolysis of GTP, the

resulting G



 GDP complex dissociates from AC and reas-

sociates with G



to reform inactive G protein. Since G



hydrolyzes its bound GTP at a characteristic rate, it func-

tions as a molecular clock that limits the length of time that

both G



 GTP and G



can interact with their effectors.

Several types of ligand–GPCR complexes may activate

the same G protein. This occurs, for example, in liver cells in

response to the binding of the corresponding hormones to

690 Chapter 19. Signal Transduction

Figure 19-16 X-ray structure of bovine rhodopsin. The protein

is viewed parallel to the plane of the plasma membrane with its

approximate position therein indicated.The protein is represented

by its transparent molecular surface with its polypeptide chain in

ribbon form colored in rainbow order from its N-terminus (blue)

to its C-terminus (red). Note its bundle of seven nearly parallel

transmembrane helices.The protein’s retinal prosthetic group

(magenta) is drawn in space-filling form as are its two Cys-linked

palmitoyl groups and its two N-linked oligosaccharide groups

(colored according to atom type with C green, N blue, O red, and

S yellow). [Based on an X-ray structure by Tetsuji Okada, National

Institute of Advanced Industrial Science and Technology, Kyoto,

Japan; and Volker Buss, University of Duisberg-Essen, Duisberg,

Germany. PDBid 1U19.]

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