From receptor to response introduction to GPCRs

All drugs that are presently on the market for clinical therapy are estimated to target less than 500 biomolecules, ranging from nucleic acids to enzymes, G protein-coupled receptors (GPCRs) and ion channels (Figure 71). Presently, GPCRs constitute one of the principal targets of drugs used in pharmacology and more than 1000 genes encoding GPCRs have been identified from human genome sequencing efforts. This represents a substantial part (± 3%) of the human genome.

A great deal of information concerning GPCRs has been acquired by investigating the P-adrenergic receptors.

Sutherland and coworkers discovered in the early sixties (Sutherland and Robison, 1966) that these receptors are able to stimulate the adenylate cyclase enzyme in isolated cell plasma membranes. Initially, it was speculated that the P-adrenergic receptor and the adenylate cyclase enzyme were parts of a single molecule. At the end of the seventies, it became clear that receptor and adenylate cyclase functions are carried by different membrane proteins and that a third, GTP-binding protein (i.e. a 'G protein'), is required to transfer the information between the receptor and the enzyme. This model is based on the discovery that guanine nucleotides such as GTP are absolutely required for adenylate cyclase stimulation and that guanine nucleotides must bind to a regulatory component in the membrane.

G proteins refer to a family of closely related membrane-associated polypeptides. By acting as a 'shuttle', they form central elements for the signal transduction between receptors and effector components (enzymes or ion channels) in the membrane. At rest, they consist of a heterotrimer (Figure 72), possessing a guanine nucleotide binding a subunit (38-52 kDa), a P subunit (35 KDa) and a Y subunit (8-10 KDa). The P and y subunits are always closely associated (i.e. P-y), and the P-y complexes are presumed to be interchangeable from one G protein to another. G proteins are not integral membrane proteins, but are anchored to the cytoplasmic face of the plasma membrane. The a subunits are predominantly hydrophilic. They are anchored to the plasma membrane due to their coupling to the P-y complexes. In addition, some of the a subunits also have a 14-carbon myristic acid added to their N-terminal domain at Gly2 (i.e. glycine located at position 2).

G Protein- Coupled Receptors: Molecular Pharmacology From Academic Concept to Pharmaceutical Research Georges Vauquelin and Bengt von Mentzer © 2007 John Wiley & Sons, Ltd. ISBN: 978-0-470-51647-8

Figure 71 Therapeutic target classes (year 2000).
Figure 72 Association of an inactive, heterotrimeric G protein to the cytoplasmic face of the cell plasma membrane.

The G protein that is required to transfer the information between the receptor and the adenylate cyclase enzyme was the first to be purified and characterized (around 1980). This G protein is now called 'Gs' (previously 'Ns'), with 's' standing for stimulatory. Since then, several additional G proteins have been discovered; the latest ones by screening DNA libraries with oligonucleotide probes. The a subunits constitute the receptor-recognizing part of the G proteins. They are also largely involved in the recognition of 'effector components' like the adenylate cyclase enzyme. The p—y complexes are not without signalling function (see later), but this is often only secondary to that of the a subunits. This explains why the identity of a G protein is determined by the identity of its a subunit.

Table 7 Principal G protein subunits and their primary effectors. Reprinted from Pharmacology and Therapeutics, 99, Hermans E., Biochemical and pharmacological control of the multiplicity of coupling at G protein-coupled receptors, 25-44, © (2003), with permission from Elsevier.



Main subtypes

Primary effector



Gas, Gaolf

Adenylate cyclase T


Ga1-1, GOj-2, GOJ-3

Adenylate cyclase 4

Ga0A, Ga0B

K+ channels T

Gat1, Gat2

Ca2+ channels 4


Cyclic GMP

Phosphodiesterase T


Gaq, Ga11, Ga14

Phospholipase C 4

Ga15, Ga16


Ga12, Ga13



ß1-5 (6?)

Different assemblies

Adenylate cyclase T/4

of P and y subunits

Phospholipases T


3-kinase T


Y1-11 (12?)

Protein kinase C T

Protein kinase D T

GPCR kinases T

Ca2+> K+ (and N+) channels

There is a striking homology between the amino acid sequences of the Ga subunits, suggesting that they have also evolved from a common ancestor. Based on the sequence of the a subunits, G proteins have been grouped into four families (Table 7):

• The Gs family includes several splice variants of as, as well as aolf (which is specifically expressed in olfactory epithelia).

• The Gi/o family consists of three distinct a; species (au, ai2 and ai3, plus splice variants) ao (which exists in two splice variants, ao1 and ao2), the two retinal transducins (at1 and at1), an a-subunit found in the gustatory epithelium (agust) and az.

• The Gq/11 family consists of aq, a11, a14, a15 and a16 (a15 and a16 appear to be the murine and human versions of the same gene).

• The G12/13 family consists of only two members, a12 and a13.

Besides the adenylate cyclase, there are many more G protein-linked effector components. They are either enzymes (e.g. guanylate cyclase, phospholipase A2 and C) or ion channels. The effector enzymes will produce second messengers including cAMP, cGMP, diacylglycerol and IP3, which in turn cause downstream effects including the opening of Ca2+ or K+ channels and the generation of other messengers, such as

Figure 73 p-adrenergic stimulation of the adenylate cyclase system.

arachidonic and phosphatidic acid. In this respect, a subunits with similar sequences often regulate the activity of the same effector systems.

Adenylate cyclase stimulation by P-adrenergic receptors illustrates the most common molecular mechanism by which G proteins transfer information from receptor to effector components in the cell membrane (Figure 73):

• In the resting state, the receptor (R), Gs and the adenylate cyclase enzyme (AC) do not interact with each other. The a subunit of Gs (i.e. as) contains tightly bound GDP.

• A messenger molecule (H) binds to the receptor to form H-R.

• H-R can now associate with Gs to form H-R-Gs. An important property of H-R-Gs is that GDP is bound less tightly and that it can be exchanged with GTP from the cytosol. In fact, the major role of the P-adrenergic receptors consists in the facilitation of the GDP/GTP exchange at the level of as.

• GTP binding disassembles the H-R-Gs complex as well as Gs itself. The complex dissociates into three parts: H-R, P-y and as which contains bound GTP (i.e. as-GTP).

• The free as-GTP (i.e. the active form of Gs) is able to associate with, and to stimulate, the adenylate cyclase enzyme. as possesses an endogenous GTP-ase activity, which is responsible for the hydrolysis of GTP into GDP (which remains inactive G protein

Pi endogenous GTP-ase catalysed by activated receptor

endogenous GTP-ase catalysed by activated receptor

active G protein

Stimulated effector component

Figure 74 Central role of G proteins.

tightly bound). This process terminates the stimulation of the adenylate cyclase enzyme.

A more general representation of this model, which clearly evidences the central role of the G proteins, is presented in Figure 74. In this model, the messenger-receptor complex facilitates the activation of the G protein (by GDP/GTP exchange and disassembling) while the endogenous GTP-ase activity of the a subunit allows the G protein to return to the basal, inactive state. Although the central 'shuttling' role of the a subunits is well established, it has also become clear over the years that some signalling functions may also be ascribed to P-y (Table 7).

Regulation (activation/inhibition) of the cyclic AMP concentration (Figure 75) and stimulation of inositol phospholipid hydrolysis (Figure 76) constitute two major mechanisms by which GPCRs affect the cell metabolism.

The adenylate cyclase enzyme was the first effector component to be discovered. It is an intrinsic protein that spans the membrane with no less than 12 hydrophobic a helixes. Nine isoforms of the mammalian adenylate cyclase have been cloned to date, and all of them are stimulated by Gs to catalyze the conversion of cytosolic ATP into the second messenger cyclic AMP and PPi. Cyclic AMP acts as an intracel-lular substitute for the chemical messenger, and it is therefore denoted as a 'second messenger'. Indeed, increased levels of cyclic AMP form the initial step in a cascade of molecular events that will give rise to the final cellular response (Figure 75). The events comprise:

• Stimulation of a specific protein kinase (protein kinase A) by cyclic AMP.

• Protein kinase A-mediated phosphorylation of a specific proteins (including the receptor itself, see later).

Ppi Cyclic Amp
Figure 75 Opposite control of the adenylate cyclase enzyme activity by Gs- and Gi- coupled receptors. ATP: adenosine triphosphate, cAMP: 3',5' cyclic AMP, PKA: protein kinase A enzyme.
Adrenergic Pkc
Figure 76 Inositol phospholipid hydrolysis and action of the hydrolysis products as second messengers. PIP2: phosphatidyl inositol 4,5-bisphosphate, IP3: inositol 1,4,5-trisphosphate, PKC: protein kinase C enzyme.
Table 8 Cellular responses mediated by cyclic AMP.

Target Tissue


Major Response


Thyroid-stimulating hormone (TSH)

Thyroid hormone synthesisand


Adrenal cortex

Adrenocorticotropic hormone (ACTH)

Cortisol secretion


Luteinizing hormone (LH)

Progesterone secretion

Muscle, liver


Glycogen breakdown



Bone resorption



Increase in heart rate and force

of contraction



Water resorption


Adrenaline, ACTH, glucagon, TSH

Triglyceride breakdown

• Altered activity/properties of the phosphorylated proteins. The nature of the phosphorylated proteins may differ from one cell type to another, so that the response will be cell-dependent (Table 8).

It has been noticed since the early eighties that several receptors do not stimulate, but rather inhibit the adenylate cyclase activity. This inhibition is also dependent on the presence of GTP and, hence, also mediated by a G protein. This inhibitory G protein has been designated as 'Gj'. In this respect, only certain isozymes of adenylate cyclase have been reported to be sensitive to ai. Since the basal cyclic AMP production is usually low in a living cell, it is not possible to observe the inhibitory effect of receptors on the adenylate cyclase activity without prior stimulation of the enzyme. This can be achieved by the simultaneous stimulation of a Gs-coupled receptor or by direct stimulation of the enzyme by forskolin.

Inositol phospholipid hydrolysis constitutes a second major mechanism by which many G protein-coupled receptors affect the cell metabolism (Figure 76). The steps involved were elucidated to great extent in the mid-eighties. The receptors recruit Gq/11 proteins to stimulate a phospholipase C enzyme (whose active site is located at the cytoplasmic side of the membrane). There are four classes of phospholipase C enzymes, called PLC-P, -y -8 and -£. From these, only the members of the PLC-P class are activated via Gq/11 proteins; the y-class is stimulated by receptor tyrosine kinases.

PLC-P cleaves 'PIP2' (phosphatidyl inositol 4,5-bisphosphate, a minor membrane phospholipid) into two compounds: diacyl glycerol and 'IP3' (inositol 1,4,5-trisphosphate). Both compounds act as 'second messengers' inside the cell:

• Diacyl glycerol remains in the membrane (since it contains the two hydrophobic fatty acid chains), but it is able to activate a cytosolic protein: protein kinase C. The activated kinase can then phosphorylate various target proteins, resulting in a modification of their activity. The action of diacylglycerol is thus very similar

0 60 120 180

time (seconds)

Figure 77 Time dependence of the cytosolic calcium concentration following the stimulation of Gq-coupled receptors (angiotensin II-stimulation in CHO cells expressing human AT1 receptors).

0 60 120 180

time (seconds)

Figure 77 Time dependence of the cytosolic calcium concentration following the stimulation of Gq-coupled receptors (angiotensin II-stimulation in CHO cells expressing human AT1 receptors).

to that of cyclic AMP, but the substrate specificity of both the kinases involved is quite different.

• On the other hand, cytosoluble IP3 will interact with specific receptors at the endoplasmic reticulum and so trigger the release of calcium from this intracellular compartment into the cytosol. The resulting increase in the cytosolic calcium concentration is only transient and can be monitored by fluorescent techniques (Figure 77). At a later stage, IP3 can be phosphorylated to IP4, which promotes the influx of extracellular calcium into the cell by opening specific calcium channels in the plasma membrane. Calcium can affect the cell metabolism on its own or via calmodulin (a soluble protein with high affinity for calcium). Calcium-calmodulin complexes can associate to other proteins in the cell, and so alter their activity.

The calcium concentration is high in the extracellular fluid (10~3 M) and in certain intracellular compartments, such as the mitochondria and the endoplasmic reticulum (= sarcoplasmic reticulum in muscle cells), but normally very low in the cytoplasm (10 ~7 M) (Figure 78). This is because calcium is continually pumped out of the cytoplasm (to the extracellular medium and in the calcium-sequestering compartments) by specific ATP-ases. The opening of small calcium-selective channels in the membrane of the endoplasmic reticulum (by IP3 receptors) as well as in the plasma membrane (by ligand- and voltage-gated channels) will allow calcium to rush down its concentration gradient, into the cytoplasm.

In general, G protein-mediated events permit an important amplification of the incoming signal (Figure 79). Indeed, a hormone- or neurotransmitter-bound receptor can stimulate many G proteins per second and a stimulated G protein may keep an effector component active for as long as 10-15 seconds. Hence, a single messenger molecule is capable of triggering the flux of a large amount of ions across the membrane

Figure 78 Ca2+ is removed from the cytoplasm by specific ATP-ases (in blue). An increase in cytoplasmic IP3 concentrationm will produce the transient opening of Ca2+ channels at the surface of the endoplasmic reticulum.
Figure 79 Signalling cascade amplification from receptor stimulation to protein kinase A activation. Black curves represent receptor occupancy.

(in the case of ion channels) or the production of a large amount of second messengers (in the case of enzymes). Such cascade-wise amplification of the signal explains the phenomenon of the 'receptor reserve', which is often encountered when comparing the degree of receptor occupation by an agonist with the evoked response.

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