1. Chapter 32 The Reception and Transmission of Extracellular Information
Biochemistry by
Reginald Garrett and Charles Grisham

2. 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?

3. 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?

4. 32.1 – What Are Hormones?

5. Figure 32.1 Nonsteroid 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.

6. (There may be others, but we doubt it...)
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

7. Figure 32.2 Structures of some steroid hormones.

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

9. 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

10. 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
intracellular catalytic domain - either a tyrosine kinase or guanylyl cyclase
Oligomeric ion channels

11. Receptors that interact with G proteins
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

12. Figure 32.5
a) The arrangement of the b2-adrenergic receptor in the membrane.
(b) Stereo image of rhodopsin, viewed parallel to the plane of the membrane. The seven-transmembrane
a-helices are indicated by roman numerals. (From Figure 2 from Palczewski,R., et al., Crystal structure of rhodopsin: A G-protein-coupled receptor. Science 289: Reprinted with permission of the AAAS.)

13. Single TMS Receptors Three 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

14. 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).

15. Shown here is fibroblast growth factor (FGF) receptor, which has three
Figure 32.6
The three classes of receptor tyrosine kinases. Class I receptors are monomeric
and contain a pair of Cys-rich repeat sequences. The insulin receptor, a typical
class II receptor, is a glycoprotein composed of two kinds of subunits in an a2b2 tetramer.
The a- and b-subunits are synthesized as a single peptide chain, together with an
N-terminal signal sequence. Subsequent proteolytic processing yields the separate
a- and b-subunits. The b-subunits of 620 residues each are integral transmembrane
proteins, with only a single transmembrane a-helix and with the amino terminus
outside the cell and the carboxyl terminus inside. The a-subunits of 735 residues
each are extracellular proteins that are linked to the b-subunits and to each other
by disulfide bonds. The insulin-biding domain is located in a cysteine-rich region
on the a-subunits. Class III receptors contain multiple immunoglobulin-like domains.
Shown here is fibroblast growth factor (FGF) receptor, which has three
immunoglobulin-like domains. (Adapted from Ulrich a., and Schlessinger,J., 1990.
Signal transduction by receptors with tyrosine kinase activity. Cell 61: )

16. 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

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

18. Soluble or Membrane-Bound
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

19. Figure 32.8 The structure of membrane-bound guanylyl cyclases.

20. 32.4 – How Are Receptor Signals Transduced?

21. Many new developments in this area
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

22. 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

23. Figure 32.10
Activation of adenylyl cyclase by heterotrimeric G proteins. Binding of hormone to
the receptor causes a conformational change that induces the receptor to catalyze
a replacement of GDP by GTP on Ga. The Ga(GTP) complex dissociates from Gbg
and binds to adenylyl cyclase, stimulating synthesis of cAMP. Bound GTP is slowly
hydrolyzed to GDP by the intrinsic GTPase activity of Ga. Ga(GDP) dissociates from
adenylyl cyclase and reassociates with Gbg. Ga and Gg are lipid-anchored proteins.
Adenylyl cyclase is an integral membrane protein consisting of 12 transmembrane
a-helical segments.

24. Figure 32.11
Adenylyl cyclase activity is modulated by the interplay of stimulatory (Gs) and inhibitory
(Gi) G proteins. Binding of hormones to b1- and b2-adrenergic receptors activates adenylyl
cyclase via Gs, whereas hormone binding to a2-receptors leads to the inhibition of adenylyl
cyclase. Inhibition may occur by direct inhibition of cyclase activity by Gia or by binding
of Gibg to Gsa.

25. Many and there may be more!
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

26. 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

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

28. Figure (a) Two views of the complex of the VC1-IIC2 catalytic domain of adenylyl cyclase and Gsa. (b) Details of the Gsa 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.)

29. 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

30. 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

31. 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

32. An oncogene and its product
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

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

34. 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

35. 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

36. Figure (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.

37. Figure The 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.

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

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

40. Figure The amino acid sequences of phospholipase C isozymes b,g, and d share two homologous domains, denoted X and Y. The sequence g-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., Role of phospholipases in generating lipid second messengers in signal transduction. The FASEB Journal 5: )

41. Ca2+ as a Second Messenger
Several sources of Ca2+ in cells!
[Ca2+] in cells is normally very low: < 1M
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

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

43. Figure 32. 22 IP3-mediated signal transduction pathways
Figure IP3-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).

44. M. Berridge's model of Ca2+ signals
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

45. Mediators of Ca2+ effects in cells
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

46. Figure (a)
Structure of uncomplexed calmodulin (blue). Calmodulin, with four Ca2+ binding domains, forms a dumbbell-shaped structure with two globular domains joined by an extended, central helix. Each globular domain juxtaposes two Ca2+-binding EF-hand domains. An intriguing feature of these EF-hand domains is their nearly identical three-dimensional structure despite a relatively low degree of sequence
homology (only 25% in some cases). (b,c) Complex of calmodulin (blue) with
a peptide from myosin light chain kinase (red); (b) side view; (c) top view.

47. Figure 32. 24 Helical wheel representations of (a) a model peptide
Figure Helical 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., How calmodulin binds its targets: Sequence independent recognition of amphiphilic a-helices. Trends in Biochemical Sciences. 15:59-64.)

48. 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

49. Figure 32. 27 The structure of a phorbol ester
Figure The structure of a phorbol ester. Long-chain fatty acids predominate at the 12-position, whereas acetate is usually found at the 13-position.

50. 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

51. 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

52. Heptadecapeptide secreted by the antral mucosa of stomach
Gastrin as an Example
Heptadecapeptide secreted by the antral mucosa of stomach
Gastrin stimulates acid secretion in stomach
Product of preprogastrin residues
Signal peptide cleavage leaves progastrin 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

53. 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

54. 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

55. 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

56. which they are found. (WW domain coordinates kindly provided by Harmut
Figure 32.30
Six of the protein modules that are found in cell-signaling proteins. Shown for each are a molecular graphic image of the module, together with primary structures of several proteins in
which they are found. (WW domain coordinates kindly provided by Harmut
Oshkinat, Forschungsinstitut für Molekulare Pharmakologie, and Marius Sudol,
Mount Sinai School of Medicine.)

57. 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!

58. 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

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

60. 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!