garrett and grisham, biochemistry, third edition chapter 32 the reception and transmission of...

Garrett and Grisham, Biochemistry, Third Edition

Chapter 32

The Reception and Transmission of Extracellular Information

Biochemistry

by

Reginald Garrett and Charles Grisham


Essential Question

• What are these mechanisms of information transfer that mediate the molecular basis of hormone action and that use excitable membranes to transduce the signals of neurotransmission and sensory systems?


Outline• What Are Hormones?• What Are Signal Transduction Pathways?• How Do Signal-Transducing Receptors Respond to the

Hormonal Message?• How Are Receptor Signals Transduced?• How Do Effectors Convert the Signals to Actions in the

Cell?• What Is the Role of Protein Modules in Signal

Transduction?


32.1 – What Are Hormones?

Figure 32.1Nonsteroid hormones bind exclusively to plasma membrane receptors, which mediate the cellular responses to the hormone. Steroid hormones exert their effects either by binding to plasma membrane receptors or by diffusing to the nucleus, where they modulate transcriptional events.


Classes of Hormones

(There may be others, but we doubt it...) • Steroid Hormones - derived from cholesterol-

regulate metabolism, salt/water balances, inflammation, sexual function

• Amino Acid Derived Hormones - epinephrine, etc.- regulate smooth muscle , blood pressure, cardiac rate, lipolysis, glycogenolysis

• Peptide Hormones - regulate many processes in all tissues - including release of other hormones

Figure 32.2Structures of some steroid hormones.

Figure 32.3The conversion of prepro-opiomelanocortin to a family of peptide hormones, including corticotropin, -and -lipotropin, - and -MSH, and endorphin.


32.3 – How Do Signal-Transducing Receptors Respond to the Hormonal

Message?• Non-steroid hormones bind to plasma

membrane and activate a signal-transduction pathway inside the cell

• Steroid hormones may either

– bind to the plasma membrane

– or

– enter the cell and travel to the nucleus


Types of Receptors

Three that we know of... • 7-transmembrane segment receptors

– extracellular site for hormone (ligand)– intracellular site for GTP-binding protein

• Single-transmembrane segment receptors– extracellular site for hormone (ligand)– intracellular catalytic domain - either a

tyrosine kinase or guanylyl cyclase • Oligomeric ion channels


7-TMS Receptors

Receptors that interact with G proteins • Seven putative alpha-helical transmembrane

segments • Extracellular domain interacts with hormone • Intracellular domain interacts with G proteins • Adrenergic receptors are typical• Note desensitization by phosphorylation,

either by ARK or by protein kinase A

Figure 32.5


Single TMS ReceptorsThree main classes

• Extracellular domain to interact with hormone • Single transmembrane segment • Intracellular domain with enzyme activity • Activity is usually tyrosine kinase or guanylyl

cyclase • Each of these has a "nonreceptor" counterpart • src gene kinase - pp60v-src was first known • Two posttranslational modifications

Figure 32.9(a) The soluble tyrosine kinase pp60v-src is anchored to the plasma membrane via an N-terminal myristyl group. (b) The structure of protein tyrosine kinase pp60v-src, showing AMP-PNP in the active site (ball-and-stick), Tyr416 (red), and Tyr527(yellow). Tyr527 is phosphorylated (purple).

Figure 32.6


Receptor Tyrosine Kinases

Membrane-associated allosteric enzymes

• How do single-TMS receptors transmit the signal from outside to inside??

• Oligomeric association is the key!

• Extracellular ligand binding

Figure 32.7Ligand (hormone)-stimulated oligomeric association of receptor tyrosine kinases.


Guanylyl Cyclases

Soluble or Membrane-Bound • Membrane-bound GCs are the other group

of single-transmembrane-segment receptors (besides RTKs)

• Peptide hormones activate the membrane forms

• Note speract and resact, from mammalian ova

• Activation may involve oligomerization of receptors, as for RTKs

Figure 32.8The structure of membrane-bound guanylyl cyclases.


32.4 – How Are Receptor Signals Transduced?


G Proteins

Many new developments in this area

• Two kinds: "heterotrimeric G proteins" and "small G proteins"

• X-ray diffraction structures for several of these are available

• Structures shed new light on possible functions


Heterotrimeric G Proteins

A model for their activity • Binding of hormone, etc., to receptor

protein in the membrane triggers dissociation of GDP and binding of GTP to -subunit of G protein

• G-GTP complex dissociates from G and migrates to effector sites, activating or inhibiting

• But it is now clear that G also functions as a signaling device

Figure 32.10

Figure 32.11


Second Messengers

Many and there may be more!• The hormone is the "first messenger" • The second messenger - Ca2+, cAMP or other

- is released when the hormone binds to its (extracellular) receptor

• The second messenger then activates (or inhibits) processes in the cytoplasm or nucleus

• Degradation and/or clearance of the second messenger is also (obviously) important


cAMP and Glycogen Phosphorylase

Earl Sutherland discovers the first second messenger

• In the early 1960s, Earl Sutherland showed that the stimulation of glycogen phosphorylase by epinephrine involved cyclic adenosine-3',5'-monophosphate

• He called cAMP a "second messenger" • cAMP is synthesized by adenylyl cyclase

and degraded by phosphodiesterase

Figure 32.12Cyclic AMP is synthesized by membrane-bound adenylyl cyclase and degraded by soluble phosphodiesterase.

Figure 32.13(a) Two views of the complex of the VC1-IIC2 catalytic domain of adenylyl cyclase and Gs. (b) Details of the Gs complex in the same orientation as the structures in (a). SW-1 and SW-2 are “switch regions,” whose conformations differ greatly depending on whether GTP or GDP is bound. (Courtesy of Alfred Gilman, University of Texas Southwestern Medical Center.)


How are the hormone receptor and AC coupled?

• Purified AC and purified receptor, when recombined, are not coupled.

• Rodbell showed that GTP is required for hormonal activation of AC

• In 1977, Elliott Ross and Alfred Gilman at Univ. of Virginia discovered a GTP-binding protein which restored hormone stimulation to AC

• Hormone stimulates receptor, which activates GTP-binding protein, which activates AC


Signaling Roles for G()

A partial list • Potassium channel proteins • Phospholipase A2 • Yeast mating protein kinase Ste20 • Adenylyl cyclase • Phospholipase C • Calcium channels • Receptor kinases


Stimulatory and Inhibitory G

G proteins may either stimulate or inhibit an effector.

• In the case of adenylyl cyclase, the stimulatory G protein is known as Gs and the inhibitory G protein is known as Gi

• Gi may act either by the Gi subunit binding to AC or by the Gi complex complexing all the Gs and preventing it from binding to AC

• Read about the actions of cholera toxin and pertussis toxin


The ras Gene and p21ras

An oncogene and its product • a gene first found in rat sarcoma virus • Normal cellular ras protein activates cellular

processes when GTP is bound and is inactive when GTP has been hydrolyzed to GDP

• Mutant (oncogenic) forms of ras have severely impaired GTPase activity, so remain active for long periods, stimulating excessive growth and metabolic activity - causing tumors to form

Figure 32.15The structure of Ras complexed with (a) GDP and (b) GMP-PNP. The Ras p21-GMP-PNP complex is the active conformation of this protein.


Phospholipases Release Second Messengers

• Inositol phospholipids yield IP3 and DAG

• PLC is activated by 7-TMS receptors and G proteins

• PLC is activated by receptor tyrosine kinases (via phosphorylation)

• Note PI metabolic pathways and the role of lithium


Other Lipids as Messengers

Recent findings - lots more to come • More recently than for PI, other

phospholipids have been found to produce second messengers!

• PC can produce C20s, DAG and/or PA • Sphingomyelin and glycosphingolipids also

produce signals • Ceramide (from SM) is a trigger of

apoptosis - programmed cell death

Figure 32.16(a) The general action of phospholiapase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). (b) The synthesis of second messengers from phospholipids by the action of phospholipases and sphingomyelinase.

Figure 32.17The family of second messengers produced by phosphorylation and breakdown of phosphatidylinositol. PLC action instigates a bifurcating pathway culminating in two distinct and independent second messengers: DAG and IP3.

Figure 32.18Phospholipase C- is activated specifically by Gq, a GTP-binding protein, and also by Ca2+.

Figure 32.19Phospholipase C- is activated by receptor tyrosine kinases and by Ca2+.

Figure 32.20The amino acid sequences of phospholipase C isozymes ,, and share two homologous domains, denoted X and Y. The sequence -isozyme contains src homology domains, denoted SH2 and SH3, SH2 domains (approximately 100 residues in length) interact with phosphotyrosine-containing proteins (such as RTKs), whereas SH3 domains mediate interactions with cytoskeletal proteins. (Adapted from Dennis,E.,Three,S., Gi8llah,M., and Hannun,E., 1991. Role of phospholipases in generating lipid second messengers in signal transduction. The FASEB Journal 5:2068-2077.)


Ca2+ as a Second Messenger

Several sources of Ca2+ in cells! • [Ca2+] in cells is normally very low: < 1M • Calcium can enter cell from outside or

from ER and calciosomes • CICR - Calcium-Induced Calcium Release

- is very, very similar to what happens at the foot structure in muscle cells!

• IP3 (made by action of phospholipase C) is the trigger

• See Figures 32.21-32.22

Figure 32.21Cytoplasmic [Ca2+] increases occur via the opening of Ca2+ channels in the membranes of calciosomes, the endoplasmic reticulum, and the plasma membrane.

Figure 32.22IP3-mediated signal transduction pathways. Increased [Ca2+] activates protein kinases, which phosphorylate target proteins. Ca2+/CaM represents calci-calmodulin (Ca2+ complexed with the regulatory protein calmodulin).


Calcium Oscillations!

M. Berridge's model of Ca2+ signals • Ca2+ was once thought to merely rise in

cells to signal and drop when the signal was over

• Berridge's work demonstrates that Ca2+ levels oscillate in cells!

• The purpose may be to protect cell components that are sensitive to high calcium, or perhaps to create waves of Ca2+ in the cell


Ca2+-Binding Proteins

Mediators of Ca2+ effects in cells • Many cellular proteins modulate Ca2+

effects • 3 main types: protein kinase Cs, Ca2+-

modulated proteins and annexins • Kretsinger characterized the structure of

parvalbumin, prototype of Ca2+-modulated proteins

• "EF hand" proteins bind BAA helices

Figure 32.23(a)

Figure 32.24Helical wheel representations of (a) a model peptide. Ac-WKKLLKLLKKLLKL-CONH2,and (b) the calmodulin-binding domain of spectrin. Positively charged and polar residues are indicated in green, and hydrophobic residues are orange. (Adapted from O’Neil,K., and DeGrado,W., 1990. How calmodulin binds its targets: Sequence independent recognition of amphiphilic -helices. Trends in Biochemical Sciences. 15:59-64.)


Transduction of two second messenger signals

PKC is activated by DAG and Ca2+

• Most PKC isozymes have several domains, including ATP-binding domain, substrate-binding domain, Ca-binding domain and a phorbol ester-binding domain

• Phorbol esters are apparent analogues of DAG

• Cellular phosphatases dephosphorylate target proteins

• Read about okadaic acid

Figure 32.27The structure of a phorbol ester. Long-chain fatty acids predominate at the 12-position, whereas acetate is usually found at the 13-position.


The Polypeptide Hormones

Common features of synthesis • All secreted polypeptide hormones are

synthesized with a signal sequence (which directs them to secretory granules, then out)

• Usually synthesized as inactive preprohormones ("pre-pro" implies at least two processing steps)

• Proteolytic processing produces the prohormone and the hormone


Proteolytic Processing

A mostly common pathway • Proteolytic cleavage of the hydrophobic N-

terminal signal peptide sequence • Proteolytic cleavage at a site defined by

pairs of basic amino acid residues • Proteolytic cleavage at sites designated by

single Arg residues • Post-translational modification: C-terminal

amidation, N-terminal acetylation, phosphorylation, glycosylation


Gastrin as an ExampleHeptadecapeptide secreted by the antral mucosa of

stomach • Gastrin stimulates acid secretion in stomach • Product of preprogastrin - 101-104 residues • Signal peptide cleavage leaves progastrin - 80-83

residues • Cleavage at Lys and Arg (basic) residues and C-

terminal amidation leaves gastrin • N-terminal residue of gastrin is pyroglutamate • C-terminal amidation involves destruction of Gly


Protein-Tyrosine Phosphatases

The enzymes that dephosphorylate Tyr-P• Some PTPases are integral membrane

proteins • But there are also lots of soluble PTPases • Cytoplasmic PTPases have N-term. catalytic

domains and C-terminal regulatory domains • Membrane PTPases all have cytoplasmic

catalytic domain, single transmembrane segment and an extracellular recognition site


Soluble Guanylyl Cyclases

Receptors for Nitric Oxide • NO is a reactive, free-radical that acts either as a

neurotransmitter or as a second messenger • NO relaxes vascular smooth muscle (and is thus

involved in stimulation of penile erection) • NO also stimulates macrophages to kill tumor cells and

bacteria • NO binds to heme of GC, stimulating GC activity 50-fold • Read about NO synthesis and also see box on Alfred

Nobel


Protein Modules in Signal Transduction

• Signal transduction in cell occurs via protein-protein and protein-lipid interactions based on protein modules

• Most signaling proteins consist of two or more modules

• This permits assembly of functional signaling complexes

Figure 32.30


Localization of Signaling Proteins

• Adaptor proteins provide docking sites for signaling modules at the membrane

• Typical case: IRS-1 (Insulin Receptor Substrate-1)

– N-terminal PH domain

– PTB domain

– 18 potential tyrosine phosphorylation sites

– PH and PTB direct IRS-1 to receptor tyrosine kinase - signaling events follow!


Signaling Pathways from Membrane to the Nucleus

• The complete path from membrane to nucleus is understood for a few cases

• See Figure 32.4• Signaling pathways are redundant• Signaling pathways converge and diverge• This is possible with several signaling

modules on a signaling protein

Figure 32.4A complete signal transduction pathway that connects a hormone receptor with transcription events in the nucleus. A number of similar pathways have been characterized.


Module Interactions Rule!

• The interplay of multiple modules on many signaling proteins permits a dazzling array of signaling interactions

• See Figure 32.30• We can barely conceive of the probable

extent of this complexity• For example, it is estimated that there are

approximately 1000 protein kinases in the typical animal cell - all signals!

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