LOGO G Protein-Coupled Signal Transmission Pathways 授課老師 : 褚俊傑副教授 ( 生物科技系暨研究所 ) 聯絡電話 : 0986-581835 電子信箱 : [email protected]

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G Protein-Coupled Signal

Transmission Pathways

授課老師 : 褚俊傑副教授 ( 生物科技系暨研究所 )聯絡電話 : 0986-581835電子信箱 : [email protected]

Gerhard KraussBiochemistry of Signal Transduction and Regulation(3rd Edition) ISBN: 3-527-30591-2

The G protein cycle.

The receptor–G-protein complex remains the only major G protein conformation for which atomic-scale structural information is unavailable. In the resting state, G proteins are heterotrimers of GDP-bound α- (blue), β- (green) and γ- (yellow) subunits (Gαt/iβ1γ1). On binding of an extracellular stimulus (light purple), receptors (pink) (such as bovine rhodopsin) undergo a conformational change that permits G protein binding and catalyses GDP release from Gα. Once GDP is released, a stable, high-affinity complex is formed between the activated receptor (R*) and G protein. Binding of GTP (green) to Gα destabilizes this complex, allowing both subunits, Gα(GTP) (Gαt(GTPγS) ND2) and Gβγ, to interact with downstream effector proteins (purple) (Gαi/q(GDP·AlF4?)·GRK2·Gβ1γ2). The signal is terminated on hydrolysis of GTP to GDP by Gα, which may be catalysed by regulator of G protein signalling (RGS) proteins (dark red) (Gαt/i(GDP·AlF4).

Outline

5.1 Transmembrane Receptors: General Structure and Classification

5.2 Structural Principles of Transmembrane Receptors

5.3 G Protein-Coupled Receptors

5.4 Regulatory GTPases

5.5 The Heterotrimeric G Proteins

5.6 Effector Molecules of G Proteins


Signal transmission into the cell interior takes place by reaction chains, in which several individual reactions generally run in sequence and involve many signal proteins. The nature of the extracellular signal can be very diverse and may include extracellular signal molecules, such as low-molecular-weight messenger substances or proteins, or sensory signals such as light signals.

The cell uses two principal ways to transduce signals into the interior of the cell. One way is exemplified by nuclear receptor signaling, where the signaling molecule crosses the cell membrane and activates the receptor in the interior of the cell. In the other major way the signal is registered at the cell membrane and transduced into the cell by transmembrane proteins.

Three different types of transmembrane proteins participate in this mode of signaling:

www.themegallery.com LOGOTransmembrane Receptors


Signaling via Transmembrane Receptors Transmission of the signal implies specific

communication with the effector protein, the next component of the signal transmission pathway on the inner side of the cell membrane. In this process enzymatic activities can be triggered and/or the activated receptor engages in specific interactions with downstream signal proteins.

An intracellular signal chain is set in motion, which finally triggers a defined biochemical response of the target cell (Fig. 5.1a). Sensory signals (light, pressure, odor, taste) can be received as well by transmembrane receptors and can be transmitted into intracellular signals.

Fig. 5.1 Mechanism of signal transduction at membranes. a) Signal transmission via ligand-controlled transmembrane receptors. The ligand L binds to the extracellular domain of a transmembrane receptor, whereby the receptor is activated for signal transmission to the cytosolic side. The cytosolic domain of the activated receptor R* transmits the signal to signal proteins next in sequence.


Signaling via Ligand-gated Ion Channels One simply designed path of signal transmission

is found in neuronal communication. Transmembrane receptors are also used for signal transmission here. These have the character of a ligand-gated ion channel (Fig. 5.1b).

Binding of a ligand (neurotransmitter or neurohormone) to the transmembrane receptor leads to a conformational change of the receptor that enables the flow of ions through the membrane.

In this case, the receptor presents itself as an ion channel with an open state controlled by ligand binding to the outer side (or also to the inner side).

Fig. 5.1 Mechanism of signal transduction at membranes. b) Signal transduction via ligand-controlled ion channels. The ligand binds to the extracellular side of a receptor that also functions as an ion channel. Ligand binding induces the opening of the ion channel, there is an ion efflux and a change in the membrane potential.


Signaling via Ligand-gated Ion Channels Another mechanism of signaling across the cell

membrane uses changes in membrane potential. A change in membrane potential induces the opening of an ion channel, and ions cross the membrane.

In this case, the change of the ion’s milieu is the intracellular signal. Ion channels with an open state regulated by changes in membrane potential are known as voltage-gated ion channels (Fig. 5.1c).

The potential-driven passage of ions through ion channels is the basis for stimulation in nerves.

Fig. 5.1 c) Signal transduction via voltage-gated ion channels. A change in the membrane potiential V is registered by an ion channel which transitions from the closed to the open state.


Intracellular Activation of Receptors We also know of transmembrane receptors for which

the reception of the signal and activation take place on the inner side of the membrane. The cGMP-dependent ion channels involved in signal conduction in the vision process are ligand-regulated ion channels with an open state controlled by intracellularly created cGMP.

Another example is the receptors for inositol triphosphate which are localized in the membrane of Ca2+ storage organelles and also have the character of ligand-controlled ion channels. Inositol triphosphate is an intracellular messenger substance that binds to the cytosolic side of the corresponding receptor located in the membrane of cell organelles.


5.2.1 The Extracellular Domain of Transmembrane Receptors

5.2.2 The Transmembrane Domain

5.2.3 The Intracellular Domain of Membrane Receptors

5.2.4 Regulation of Receptor Activity


Transmembrane receptors are integral membrane proteins, i.e., they possess a structural portion that spans the membrane. An extracellular domain, a transmembrane domain and an intracellular or cytosolic domain can be differentiated within the structure (Fig. 5.2a).

Fig. 5.2 Structural principles of transmembrane receptors. a) Representation of the most important functional domains of transmembrane receptors.

5.2.1 The Extracellular Domain of Transmembrane Receptors

In many receptors, the extracellular domain contains the ligand-binding site. Glycosylation sites, i.e. attachment sites for carbohydrate residues, are also located nearby in the extracellular domain.

The extracellular localized protein portion may be formed from a continuous protein chain and may include several hundred amino acids. If the receptor crosses the membrane with several transmembrane segments, the extracellular domain is formed from several loops of the protein chain that may be linked by disulfide bridges.

Transmembrane receptors may show homotrophic composition (identical subunits) or heterotrophic composition (different subunits, Fig. 5.2b), so that the extracellular domain may be made up of several identical or different structural elements.

Fig. 5.2 Structural principles of transmembrane receptors. b) Examples of subunit structures. Transmembrane receptors can exist in a monomeric form (1), dimeric form (2) and as higher oligomers (3,4). Further subunits may associate at the extracellular and cytosolic domains, via disulfide bridges (3) or via non-covalent interactions (4).


The transmembrane domains have different functions, according to the type of receptor. For ligand-controlled receptors, the function of the transmembrane domain is to pass the signal on to the cytosolic domain of the receptor. For ligand-controlled ion channels, the transmembrane portion forms an ion pore that allows selective and regulated passage of ions.

The transmembrane receptors span the 5 nm thick phospholipid bilayer of the cell membrane with structural portions known as transmembrane elements. The inner of a phospholipid layer is hydrophobic and, correspondingly, the surface of the structural elements that come into contact with the inner of the phospholipid double layer also has hydrophobic character.


Structure of Transmembrane Elements High-resolution structural information about the

transmembrane elements of transmembrane receptors could recently be obtained on the example of rhodopsin, the light-activated G protein coupled receptor of the vision process (Fig. 5.3).

These data, together with earlier data on the structures of other transmembrane proteins (e.g., bacteriorhodopsin), have confirmed that -helices are the principal structural building blocks of the transmembrane elements of membrane receptors.

Fig. 5.3 Three-dimensional structure of rhodopsin. Two views of rhodopsin. A) The seven -helices of the G protein-coupled receptor rhodopsin weave back and forth through the membrane lipid bilayer (yellow lines) from the extracellular environment (bottom) to the cytoplasm (top). The chromophore 11-cis retinal (yellow) is nested among the transmembrane helices .B) View into the membrane plane from the cytoplasmic side of the membrane. Roman numerals indicate numbered helices.


Structure of Transmembrane Elements

In addition to a-helices, proteins also use -structures to cross the membrane. The transmembrane domain of the bacterial OmpF porin is made up of -elements (see Fig. 5.4). The -elements, in this case, are not mostly made up of hydrophobic amino acids and form a barrel-like structure.

Fig. 5.4 The OmpF porin from Eschericia coli is an integral membrane channel-forming protein which spans the outer membrane in Gram-negative bacteria. The structure of a monomer of the OmpF porin is shown. In total, 16 -bands are configured in the form of a cylinder and form the walls of a pore through which selective passage of ions takes place.

5.2.3 The Intracellular Domain of Membrane Receptors

Two basic mechanisms are used for conduction of the signal to the inner side of the membrane (Fig. 5.5):

Starting from the activated receptor, a large number of reactions can be set in motion (Fig. 5.5). One main route of signal transmission takes place by activation of G proteins, another via activation of tyrosine-specific protein kinases, and a further route is via activation of ion channels. In the further course of G protein-mediated signal transmission, secondary diffusible signals are often formed: the “second messenger” molecules (see Chapters 3 and 6).

Fig. 5.5 General functions of transmembrane receptors. Extracellular signals convert the transmembrane receptor from the inactive form R to the active form R*. The activated receptor transmits the signal to effector proteins next in the reaction sequence. Important effector reactions are the activation of heterotrimeric G-proteins, of protein tyrosine kinases and of protein tyrosine phosphatases. The tyrosine kinases and tyrosine phosphatases may be an intrinsic part of the receptor or they may be associated with the receptor. The activated receptor may also include adaptor proteins in the signaling pathway or it may induce opening of ion channels.


The cell has various mechanisms available, with the help of which the number and activity of transmembrane receptors can be regulated. The aim of regulation is, for example, to weaken signal transmission via the receptor during conditions of long-lasting hormonal stimulation.

Furthermore, signal transduction by transmembrane receptors may be modulated via crosstalk with other signaling pathways. The structural elements involved in regulation of receptor activity are generally located in the cytosolic domain.


These are, above all, protein sequences that permit phosphorylation of the receptor by protein kinases. Phosphorylation at Ser/Thr or Tyr residues of the cytosolic domain may lead to inactivation or activation of the receptor and thus weaken or strengthen signal transmission.

In this way, Ser/Thr-phosphorylation is used in the process of internalization of receptors in order to remove the receptor from circulation after it has been activated (see Section 5.3.4). The protein kinases involved are often part of other signaling pathways and can link the activity of the transmembrane receptors to other signaling networks.


5.3.1 Structure of G Protein-Coupled Receptors

5.3.2 Ligand Binding

5.3.3 Mechanism of Signal Transmission

5.3.4 Switching Off and Desensitization of 7-Helix Transmembrane Receptors

5.3.5 Dimerization of GPCRs


Of the transmembrane receptors that receive signals and conduct them into the cell interior, the G protein-coupled receptors form the largest single family. About 5% of the genome of the worm Caenorhabditis elegans is occupied by genes encoding G protein-coupled receptors.

In vertebrates, more than 1000 different G protein-coupled receptors are found that may be activated by extracellular ligands or sensory signals. The ligands include biogenic amines, such as adrenaline and noradrenaline, histamine, serotonin, lipid derivatives, nucleotides, retinal derivatives, peptides such as bradykinin and large glycoproteins such as luteinizing hormone, and parathormone (see also Table 3.1).

Tab. 5.1 Classification of the heterotrimeric G proteins according to the α-subunits.



Examplary for the G-protein-coupled receptors, Fig. 5.6 shows the two dimensional model of bovine rhodopsin. The three-dimensional structure of rhodospsin has been directly visualized high resolution X-ray analysis that shows a bundle of 7 transmembrane helices, as predicted from a multitude of biochemical and biophysical studies.

This structure provides a frame upon which the 3D-structure of the huge family of 7-helix transmembrane receptors can be modeled. Sequence comparisons, biochemical and biophysical data indicate that the transmembrane bundle structure is conserved among GPCRs and possibly within the entire GPCR family.

By contrast, the loops and termini are more divergent in amino acid sequence and possibly in three-dimensional structure.

Fig. 5.6 Two dimensional model of rhodopsin. The extracellular (intradiscal) and intracellular regions of rhodopsin each consist of three interhelical loops (given the prefixes E(extracellular)-I to E-III or C(cytoplasmic)-I to C-III. A conserved disulfidebridge is found on the extracellular side linking EII with E-III. On the intracellular side, a short helix runs parallel to the membrane surface. In the native protein, the C-terminus carries two palmitoylated Cys-residues which function as membrane anchorscausing formation of a putative fourth intracellular loop.


The only structural feature common to all G protein-coupled receptors is the presence of the seven transmembrane helices connected by alternating extracellular and intracellular loops, with the amino terminus located on the extracellular side and the carboxy terminus on the intracellular side. Apart from that feature, the overall sequence homology among the G protein-coupled receptors is low.

Significant sequence homology is found, however, within three subfamilies, designated family A, B and C receptors. The classification is based on the size of the extracellular loops, the presence of key residues and the formation of disulfide bonds (Fig. 5.7). Family A includes the rhodopsin/-adrenergic receptor, family B includes calcitonin receptors and family C includes receptors for c-amino butyric acid, Ca2+ and glutamate.

Fig. 5.7 Classification of GPCRs. The G protein-coupled receptors can be divided intothree major subfamilies (see Gether, 2001)Family A receptors are characterized by a series of highly conserved key residues (black letter in white circles). In most family A receptors, a disulfide bridge is connecting the E-II and E-III loops. In addition, a majority of the receptors have a palmitoylated cysteine in the cytoplasmic C-terminus. Ligands include the biogenic amines (adrenaline, serotonine, doapmine, histamine), neuropetide Y, adenosine, chemokines and melatonine, among others.

Fig. 5.7 Classification of GPCRs. The G protein-coupled receptors can be divided intothree major subfamilies (see Gether, 2001)Family B receptors are characterized by a long extracellular N-terminus containing a series of cysteine residues presumably forming a network of disulfide bridges. Representative members of the family B receptors include calcitonine receptor, glucagon receptor and parat hormone receptors.

Fig. 5.7 Classification of GPCRs. The G protein-coupled receptors can be divided intothree major subfamilies (see Gether, 2001)Family C receptors are characterized by a very long N-terminus forming the extracellular ligand binding site. There is only one putative disulfide bridge and the third cytoplasmic loop is very small. The taste receptors, the metabotropic glutamate receptors, the c-aminobutyric acid (GABA) receptors and Ca2+-receptors belong to this class, among others.

5.3.2 Ligand Binding

The area of ligand binding has been particularly well defined for the receptors of classical “small ligands” (adrenaline, noradrenaline, dopamine, serotonine, histamine). Targeted mutagenesis, biochemical, biophysical and pharmacological investigations have shown that these ligands are bound in a binding crevice formed by the transmembrane helices.

In agreement with this model, it has been shown that the extracellular and intracellular sequence portions of the receptors are not needed for ligand binding in these cases. Rhodopsin is a unique case, since the retinal ligand is covalently attached by Schiff-base linkage to a Lys residue of transmembrane helix VII. In that case too, the binding site is deeply buried in the interior of the transmembrane segment (see Fig 5.3).

5.3.3 Mechanism of Signal Transmission

The heterotrimeric G protein, which exists as the inactive GDP form, now binds via its - and possibly γ-subunit to the activated receptor and is activated itself. An exchange of GDP for GTP takes place, and the βγ-subunit of the G protein dissociates (see Section 5.5.3). Once the G protein is activated, it frees itself from the complex with the receptor, which either returns to its inactive ground state or activates further G proteins.


A phenomenon often seen in transmembrane receptors in general, and in G protein coupled receptors in particular, is desensitization (Fig.5.8). Desensitization means a weakening of the signal transmission under conditions of long-lasting stimulation by hormones, neurotransmitters or sensory signals.

Despite the persistent effect of extracellular stimuli, the signal is no longer passed into the cell interior, or only in a weakened form, during desensitizing conditions. This is a mechanism with which both short-term and long-term regulation of receptor activity is possible.

Fig. 5.8 General principle of desensitization of G-protein-coupled receptors. Desensitization of a hormone-bound receptors can take place by two principle routes, schematically represented in the figure. A suppressing influence may be exerted on the receptor system via proteins (X) of a signal chain, triggering inhibition of the signal chain. Receptor systems may also mutually influence one another in that a signal protein X formed in one signal chain mediates the desensitization of another receptor system R*, and vice versa.


Phosphorylation by cAMP-dependent protein kinases (Fig. 5.8)

Phosphorylation of the cytoplasmic domain of 7-helix transmembrane receptors can take place via cAMP-dependent protein kinases (protein kinase A) or via protein kinase C (Chapter 7) (Fig. 5.9). This is a feedback mechanism. The hormonal activation of the receptor leads, via G proteins and adenylyl cyclase/cAMP, to activation of protein kinases of type A (see Sections 5.6.1 and 6.1, and Chapter 7).

The activated protein kinases phosphorylate the receptor in the region of the cytoplasmic domain on Ser/Thr residues. Regulation via adenylyl cyclase/cAMP/proteinkinase A is an example of a heterologous desensitization, since adenylyl cyclase can be activated by a variety of signals originating from different signaling pathways (see Section 5.6.1).

Fig. 5.9 Desensitization of G-protein-coupled receptors via cAMP-dependent protein kinases. Starting from an activated receptor R*, the signal is transmitted via the G-subunit of the G-protein to adenylyl cyclase. The latter is activated and forms cAMP. This activates a protein kinase of type A that passes the signal in the form of a Ser/Thr-specific protein phosphorylation to substrate proteins. One of the substrates is also the receptor that is phosphorylated in the region of the cytoplasmic domain by the activated protein kinase A. The ligand-bound receptor is preferentially phosphorylated. As a consequence of phosphorylation, activation of further G-proteins by the receptor is suppressed.


Phosphorylation via G protein coupled receptor protein kinases (GRK)

The major mechanism for the homologous desensitization of agonist-bound 7-helix transmembrane receptors consists of a two-step process in which the agonist-bound receptor is phosphorylated by a GRK and then binds an arrestin protein which interrupts signaling to the G protein. Receptor phosphorylation by GRKs triggers several reactions (Fig. 5.10)

Fig.5.10Receptor desensitization: phosphorylation, arrestin binding and internalization. The activated, agonist-bound receptor is phosphorylated on the cytoplasmic region by a G protein-coupled receptorprotein kinase (GRK). The phosphate residues serve as attachment sites for -arrestin which has protein kinases of the MAPK cascade associated. This serves as a trigger for internalization of the receptor to endosomes. The receptor may now be dephosphorylated and transported back to the cellmembrane (not shown in the figure).


Binding of Arrestin

For some receptors, arrestin binding serves to activate a protein kinase cascade, the MAPK cascade. In that signaling mode, arrestins function as scaffolding proteins that help to organize the three protein kinases of the MAPK pathway into a cascade of sequentially acting protein kinases delivering a signal to the level of, e.g., transcription factors (see Chapter 10).

In view of this observation, arrestins appear to function not only as “signal terminators” but rather also as activators of another signaling pathway, that of the MAPKs, providing another example of a crosstalk between different signaling pathways (Fig. 5.11).

Fig. 5.11 Activation of the MAPK cascade via -arrestin and G protein-coupled receptors. A complex is formed between arrestin and the various components of a MAPK module (see Chapter 10) in the cytosol. This multiprotein complex translocates to the plasma membrane bound receptor following ligand binding. -arrestin functions as a scaffold for the MAPK module and promotes internalization of the whole complex. This leads to generation of active MAPK and stimulation of transcription. MAPK: mitogen activated protein kinase, MEK: MAPK/ERK kinase, MEKK: MEK kinase.


Dimerization has been shown to alter the ligand-binding, signaling and trafficking properties of G protein-coupled receptors.

In addition to homodimers, the formation of heterodimers with related members of the same subfamily has been also reported. The structural, functional and mechanistic consequences of the formation of the oligomeric receptor complexes remain to be elucidated.


Cross-talk between G protein—coupled receptors (GPCRs), ligand-gated ion channels (LGChs) and receptor protein tyrosine kinases (RTKs).


5.4.1 The GTPase Superfamily: General Functions and Mechanism

5.4.2 Inhibition of GTPases by GTP Analogs

5.4.3 The G-domain as Common Structural Element of the GTPases

5.4.4 The Different GTPase Families


The heterotrimeric G proteins, the major effector proteins of the 7-helix transmembrane receptors, belong to the large family of regulatory GTPases ; these bind GTP and hydrolyze it, thereby functioning as a switch in central cellular processes.

The family of regulatory GTPases is also called the GTPase superfamily.

5.4.1 The GTPase Superfamily: General Functions and Mechanism

The Switch Function of the GTPases

The regulatory GTPases are involved in signaling chains by functioning as a switch. Incoming signals are received by the GTPases and are passed on to downstream components of the signaling chain. The switch function is based on a cyclical, unidirectional transition between an active, GTP-bound form and an inactive, GDP-bound form (Fig. 5.12).

The binding of GTP brings about the transition into the active form. Hydrolysis of the bound GTP by an intrinsic GTPase activity converts the protein into the inactive, GDP-bound form.

Fig. 5.12 The switch function of the regulatory GTPases. The GTP form of the regulatory GTPases represents the “switched on” form of the GTPase, the GDP form, in contrast, the “switched off” form. The switch function of the regulatory GTPases may be controlled by guanine nucleotide exchange factors, by GTPase activating proteins (GAPs) and by G-nucleotide dissociation inhibitors (GDIs). The regulatory GTPases run through a GTPase cycle which signals flow into via GEFs and are conducted further in the form of the GTPase-GTP complex to effector molecules further down the sequence. Hydrolysis of the bound GTP ends the activated state. The rate of GTP hydrolysis is either intrinsically determined or may be accelerated via GAPs.


Regulatory GTPases have a common mechanism that enables them to switch a signal transduction chain on and off.

5.4.2 Inhibition of GTPases by GTP Analogs

Nonhydrolyzable GTP analogs are an indispensable tool in the identification and structural and functional characterization of GTPases. The GTP analogs shown in Fig. 5.13, GTPγS, β,γ-methylene GTP and β,γ-imino GTP, are either not hydrolyzed by GTPases or only very slowly. Addition of these analogs fixes the G protein in the active form; it is permanently “switched on”. For cellular signal transduction, this means permanent activation of the signal transmission pathway.

In many cases, a role of G proteins in a signal chain was inferred from the observation that nonhydrolyzable GTP analogs bring about a lasting activation of signal transmission. The GTP analogs were equally important for structural determination of the activated form of GTPases.

Fig. 5.13 Examples of non-hydrolysable GTP analogs.

5.4.4 The Different GTPase Families

The superfamily of GTPases, with over 100 members, is subdivided according to sequence homologies, molecular weight and subunit structure into further (super)families.

These are the families of the heterotrimeric G proteins, the Ras superfamily of small GTPases and the family of initiation and elongation factors (Fig. 5.15).

Fig. 5.15 The GTPase superfamily.


5.5.1 Classification of the Heterotrimeric G Proteins

5.5.2 Toxins as Tools in the Characterization of Heterotrimeric G Proteins

5.5.3 The Functional Cycle of Heterotrimeric G Proteins

5.5.4 Structural and Mechanistic Aspects of the Switch Function of G Proteins

5.5.5 Structure and Function of the βγ-Complex

5.5.6 Membrane Association of the G Proteins

5.5.7 Regulators of G Proteins: Phosducin and RGS Proteins


Regulatory cycle of heterotrimeric G proteins, for abbreviations


A common structural feature of the G proteins is their construction from three subunits (Fig. 5.16), a large α-subunit of 39–46 kDa, a β-subunit of 36 kDa and a γ-subunit of 8 kDa. The -subunit has a binding site for GTP or GDP and carries the GTPase activity.

The β- and γ-subunits exist as a tightly associated complex and are active in this form. All three subunits show great diversity, so that at least 20 different genes for α-subunits, 5 for β-subunits and 12 for γ-subunits are known in mammals. Some G protein are ubiquitous, whereas others only occur in specialized tissue.

Fig. 5.16 Structure and activation of the heterotrimeric G-proteins. Reception of a signal by the receptor activates the G-protein, which leads to exchange of bound GDP for GTP at the a-subunit and to dissociation of the βγ-complex. Further transmission of the signal may take place via Gα-GTP or via the βγ-complex, which interact with corresponding effector molecules. The α- and γ-subunits are associated with the cell membrane via lipid anchors. Signal reception and signal transmission of the heterotrimeric G-proteins take place in close association with the cell membrane. This point is only partially shown in the figure.

5.5.1 Classification of the Heterotrimeric G Proteins

Most functions of signal transmission by G proteins are realized by the a-subunit. Since different G proteins interact with very different partners, there are significant differences in the structure of the -subunits.

Based on comparison of the amino acid sequences, the Gproteins are divided into four families, the Gs, Gi, Gq and G12 families.

These families are summarized in Table 5.1, together with representative members and their characteristic properties.

Tab. 5.1 Classification of the heterotrimeric G proteins according to the α-subunits.

5.5.2 Toxins as Tools in the Characterization of Heterotrimeric G Proteins

Two bacterial toxins, namely pertussis toxin and cholera toxin, were of great importance in determining the function of G proteins.

Both toxins catalyze ADP ribosylation of proteins. During ADP ribosylation, an ADP-ribose residue is transferred from NAD+ to an amino acid residue of a substrate protein (Fig. 5.17).

Fig. 5.17 ADP-ribosylation of the G-subunit of transducin by cholera toxin. Cholera toxin catalyzes the ADP-ribosylation of the -subunit of the G-protein transducin. During the reaction, the ADP ribose residue of NAD+ is transferred to Arg174 of G,t, which inactivates the GTPase activity of G,t.

5.5.3 The Functional Cycle of Heterotrimeric G Proteins

Signal transmission via G proteins takes place in close association with the inner side of the cell membrane. Both the -subunit and the βγ-complex are associated with the membrane via membrane anchors (see Section 5.5.6).

Like all regulatory GTPases, the heterotrimeric G proteins run through a cyclical transition between an inactive, GDP-bound form and an active, GTP-bound form. Thereby, the activated G protein-coupled receptor functions as a nucleotide exchange factor, GEF. Figure 5.18 sketches the different functional states and the role of the individual subunits.

Fig. 5.18 Functional cycle of the heterotrimeric Gproteins. a) The G-proteins exist in the ground state as a heterotrimeric complex (GαGDP) · (βγ). b) The activated receptor binds to the inactive heterotrimeric complex of the G-protein and leads to dissociation of the bound GDP and the βγ-complex. c) Binding of GTP to the “empty” Gα-subunit transforms the latter into the active GαGTP state. GαGTP interacts with an effector molecule in the sequence E1 and activates the latter for further signal transmission. The released βγ-complex may also take part in signal conduction by binding to a corresponding effector molecule E2 and activating the latter for further signal conduction. d) Hydrolysis of the bound GTP terminates the signal transduction via the α-subunit.

5.5.4 Structural and Mechanistic Aspects of the Switch Function of G Proteins

A structural model of the trimeric G protein and the receptor is presented in Fig. 5.19. In this model, the known structures of the ground state of rhodopsin and the structures of the transducin Gtα· GDP · (βγ) complex have been modeled, taking into account the location of the lipid anchors and the known interaction sites between the receptor and the G protein.

Fig. 5.19 Model of the assembly of rhodopsin and transducin at the cell membrane (from Hamm, 2001). Models are based on the crystal structure and are to scale. The C-terminal residues after S316 are not shown. The orientation of transducin (Gt,α, Gt,βγ) with respect to rhodospin and the membrane is based on the charge and hydrophobicity of the surface, the known rhodosin binding sites on transducin and the sites of lipidation of Gt,α, and Gt,βγ.

5.5.5 Structure and Function of the βγ-Complex

Structures of the βγ-complex could be obtained both in the free state and in the Gα-GDP-bound state. The βγ-complex has an interesting configuration of seven b-sheet structures for the β-subunit.

5.5.6 Membrane Association of the G Proteins

Signal transmission via G proteins is inseparably linked with their membrane association.

The preceding reaction partners are transmembrane proteins, and the subsquent effector molecules, such as adenylyl cyclase, are either also transmembrane proteins or are associated with the membrane (Fig. 5.23).

Fig. 5.23 Membrane anchor of the heterotrimeric G-proteins. The lipid anchoring in the system of G-protein- coupled receptors and the corresponding G-proteins is shown. In the figure, it is assumed that the lipid anchors are located in the membrane. A possible involvement of the lipid anchor in protein interactions is not shown. The G-protein coupled receptor carries a palmitoic acid anchor at the C-terminus. The α-subunit of the heterotrimeric G-protein is associated with the membrane via a myristoic acid anchor at the N-terminus, whilst the γ-subunit of the βγ-complex uses a prenyl residue as a membrane anchor.

5.5.7 Regulators of G Proteins: Phosducin and RGS Proteins

The regulation is mostly of a negative, suppressing character and serves two purposes in particular. Firstly, the cell must try to weaken the cytoplasmic answer under conditions of persistent activation of the receptor. Secondly, the cell needs mechanisms to rapidly terminate the signal. Typically, the rate of GTP hydrolysis of the a-subunit is very slow, about 4 min–1.

The cell must be able to shorten the associated long lifetime of the activated state in a regulatable way. The most important regulatory attack points at the level of the G proteins and their receptors are (Fig. 5.24)

Fig. 5.24 Regulation of G protein-coupled receptors and of G proteins. Signal transduction by activated G protein-coupled receptors (GPCRs) is mainly regulated by phosphorylation via GPCR kinases (GRKs) leading to downregulation and desensitization. Signaling by GαGTP can be negatively controlled by regulators of G protein signaling (RGS) and by the effector proteins themselves. A negative control of βγ-complex signaling is mediated by phosducin.

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LOGO G Protein-Coupled Signal Transmission Pathways 授課老師 : 褚俊傑副教授 ( 生物科技系暨研究所 ) 聯絡電話 : 0986-581835 電子信箱 : [email protected]