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G-Protein Coupled Receptors (GPCRs) Lectures: February 28, March 2, 7, 9 and 11, 2005 ; Michael Greenwood ([email protected]). Nature Reviews Molecular Cell Biology 3 , 639-650 (2002) SEVEN-TRANSMEMBRANE RECEPTORS. Kristen L. Pierce, Richard T. Premont & Robert J. Lefkowitz

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G-Protein Coupled Receptors (GPCRs)Lectures: February 28, March 2, 7, 9 and 11, 2005;

Michael Greenwood ([email protected])

Nature Reviews Molecular Cell Biology3, 639-650 (2002)


Kristen L. Pierce, Richard T. Premont & Robert J. Lefkowitz

The Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Box 3821, Duke University Medical Center, Durham, North Carolina, 27710, USA.

Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors,

are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and

activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

This is the main review for the GPCR lectures.

Most advanced textbooks (i.e.Mol. Biol. of the Cell) cover the basics of GPCRs

Lecture topics:A. GPCRs as receptors

1-Basic structure

-7 transmembrane topology

-receptors are associated with a heterotrimeric G-protein

2- Diversity of the GPCR gene family

-3 subfamilies

-GPCRs mediate the effects of a large variety of different agonists

-multiple receptors recognize the same ligand

3- Receptor activation of heterotrimeric G-proteins

-model of GPCR activation

-different G-proteins activate distinct signalling pathways

-receptor specificity

-diversity of GPCR signalling

-G-protein independent GPCR signalling

4- Functional domains on the GPCR

-receptor pharmacology

-G-protein activating domains

-ligand binding pocket or domains

5- Inactivation of GPCR mediated signalling


-receptor desensitization at the molecular level: GRKs and arrestins

-inactivation of the receptor activated heterotrimeric G-protein by RGSs

6- GPCR dimerization

7- Alternative function of GPCRs

B. Topics in GPCR biology (if time permits)

1-Odorant receptors

2-GPCRs as drug targets: orphan and known GPCRs

3-Agonist independent activation of Angiotensin II receptors in the heart by stretch

4-Chemokine receptors: cancer metastasis; molecular piracy by virally encoded GPCRs; as entry points for HIV and malaria

1. Basic structure:

- 7 Transmembrane Domains (TMDs),

3 intracellular loops, 3 extracellular loops, N- and C-teminals

Fig. 1. Schematic representation of the membrane topology of the human β2 adrenergic receptor. The localizations of TMHs in the human β2-adrenoceptor are indicated (black lines). The core and water-lipid interface regions of the lipid membrane are indicated with light gray and dark gray colors on the background.

Each of the 7 TMHs have one characteristic residue (black circles with white text), which is found among the majority of family 1 (also called A) GPCRs.

Pharmacol Ther. 2004 Jul;103(1):21-80

1. Basic structure

Cysteine bridges

Two-dimensional topology of the human CCR5 sequence. Membrane topology of CCR5 with the extracellular space at the top and the intracellular space at the bottom. Amino acids shown to be critical for CCR5 function are highlighted by filled circles. The grey box marks the approximate position of the membrane bilayer.

Cell Signal. 2004 Nov;16(11):1201-10

1. Basic structure: GPCRs are lipoproteins

Schematic representation of a family A receptor in the cell membrane based on the packing arrangement of TMHs observed in the most recent crystal structure of rhodopsin (pdb code 1L9H). Putative TMHs are depicted as cylinders.

Pharmacol Ther. 2004 Jul;103(1):21-80

1. Basic Structure

Coupled to heterotrimeric G-protein

(A) GPCRs have a central common core made of seven transmembrane helices (TM-I to -VII) connected by three intracellular (i1, i2, i3) and three extracellular (e1, e2, e3) loops.

(B) Illustration of the central core of rhodopsin. The core is viewed from the cytoplasm. The length and orientation of the TMs are deduced from the two-dimensional crystal of bovine and frog rhodopsin (Unger et al., 1997).

The EMBO Journal (1999) 18, 1723–1729

Classification and diversity of GPCRs. (A) Three main families (1, 2 and 3) can be easily recognized when comparing their amino-acid sequences. Receptors from different families share no sequence similarity, suggesting that we are in the presence of a remarkable example of molecular convergence. Family 1 contains most GPCRs including receptors for odorants. Group 1a contains GPCRs for small ligands including rhodopsin and β-adrenergic receptors. The binding site is localized within the seven TMs. Group 1b contains receptors for peptides whose binding site includes the N-terminal, the extracellular loops and the superior parts of TMs. Group 1c contains GPCRs for glycoprotein hormones. It is characterized by a large extracellular domain and a binding site which is mostly extracellular but at least with contact with extracellular loops e1 and e3. Family 2 GPCRs have a similar morphology to group Ic GPCRs, but they do not share any sequence homology. Their ligands include high molecular weight hormones such as glucagon, secretine, VIP-PACAP and the Black widow spider toxin, α-latrotoxin. Family 3 contains mGluRs and the Ca2+ sensing receptors. Last year, however, GABA-B receptor and a group of putative pheromone receptors coupled to the G protein Go (termed VRs and Go-VN) became new members of this family. (B) Family 4 comprises pheromone receptors (VNs) associated with Gi. Family 5 includes the 'frizzled' and the 'smoothened' (Smo) receptors involved in embryonic development and in particular in cell polarity and segmentation. Finally, the cAMP receptors (cAR) have only seen found in D.discoïdeum but its possible expression in vertebrate has not yet been reported.

2. Diversity of the GPCR superfamily

The EMBO Journal (1999) 18, 1723–1729

2. Diversity….

-large variety of different agonists

2. Diversity… Multiple GPCRs can bind a single agonist: serotonin.

Serotonin (5-hydroxytryptamine or 5-HT) is involved in mediating a large number of different responses and diseases. These are now seven sub-families of 5HT receptors, 5-HT1–7, comprising a total of 14 structurally and pharmacologically distinct mammalian 5-HT receptor subtypes.

Fig. 1. Dendrogram showing the evolutionary relationship between various human 5-HT receptor protein sequences (except 5-HT5A and 5-HT5B receptors which are murine in origin).

2. Diversity….

Fig. 1. Graphical representation of the current classification of 5-HT receptors. Receptor subtypes represented by coloured boxes and lower case designate receptors that have not been demonstrated to definitively function in native systems. Abbreviations: 3′-5′ cyclic adenosine monophosphate (cAMP); phospholipase C (PLC); negative (−ve); positive (+ve).

Pharmacol Biochem Behav. 2002 Apr;71(4):533-54

2. Diversity…

GPCR subfamilies: multiple

receptors often recognize the

same ligand

TABLE 2. Examples of specificity and multiplicity of peptide ligand-receptor interactions

Endocrinology Vol. 145, No. 6 2645-2652

3. Receptor activation of heterotrimeric G-proteins.

Basic model

Hollinger et al. 2000Pharmacological Reviews

3. Receptor activation…

Simplified Model of GPCR activation

A schematic representation of how the two-state receptor model relates to the action of drugs as strong agonists, partial agonists, neutral competitive antagonists, inverse agonists, and inverse partial agonists. The inactive and active receptor conformations (R and R*, respectively) are in constant equilibrium. A strong agonist binds selectively to R*, driving the equilibrium between R and R* in favour of R*, resulting in enhanced response. A partial agonist has higher affinity for R* than for R, but with less selectivity than the strong agonist. The neutral competitive antagonist binds with equal affinity to both R and R*, so that it does not disturb the resting equilibrium and therefore does not alter basal response. An inverse strong agonist binds selectively to R, driving the equilibrium between R and R* in favour of R, resulting in decreased response, that is, when there is significant constitutive activity (basal response). An inverse partial agonist has higher affinity for R than for R*, but with less selectivity than the strong inverse agonist

British Journal of Clinical Pharmacology57 (4), 373-387.

3. Receptor activation

Allosteric model of

GPCR activation


Dissecting the allosteric two-state model. The allosteric two-state model cube.

3. Receptor activation…

GPCRs activate different

sub-classes of heterotrimeric

G-proteins and effector systems

3. Receptor activation…

GPCRs activate different sub-classes of heterotrimeric

G-proteins and effector systems (cont’d)

Nature Reviews Molecular Cell Biology3; 639-650

3. Receptor activation…

Activation of cAMP responses by Gs coupled GPCRs

How gene transcription is activated by a rise in cyclic AMP concentration.

Molecular Biology of the Cell

3. Receptor activation…

Activation of phospholipase Cβ by Gq coupled GPCRs

The hydrolysis of PI(4,5)P2 by phospholipase C-b.

Molecular Biology of the Cell

3. Receptor activation…

Receptor switching

Possible mechanism underlying the "switch" of the functional coupling of a given receptor with distinct G-proteins. Stimulation of the ‘naïve’ receptor favours the coupling with a subset of G-proteins, resulting in the activation of a preferential signalling cascade (Response A). This response includes the activation of a protein kinase that may phosphorylate the receptor and thereby progressively impair the coupling with this subset of G-proteins. In contrast, while response A is progressively inhibited, the coupling of the phosphorylated receptor with another subset of G-proteins is maintained or even enhanced, leading to the emergence of another signalling cascade (Response B).

Pharmacol Ther. 2003 Jul;99(1):25-44

3. Receptor activation…

GPCRs are unfaithful to G proteins

How can such interactions

be characterized? and/or


Two examples of transduction triggered via a direct interaction of GPCRs

with proteins containing PDZ and EVH-like domains.

3. Receptor activation…

Receptor independent activation of heterotrimeric G-Proteins



Signal regulator that influences the transfer of signal from receptor to G-protein or directly regulates the activation state of G-proteins.

Biol Cell. 2004 Jun;96(5):369-72.

3. Receptor activation…

Multiple receptors activate the same G-protein

3. Receptor activation…

Complexity of GPCR signalling


GPCRs cross talk with Receptor

Tyrosine Kinases (RTK)

Given such a diversity in

responses, how does GPCR

signaling specificity occur???

Multiple physiological responses

3. Receptor activation…Yeast as a model system to study GPCR structure, function and receptor specificity

Saccharomyces cerevisiae

Budding yeast

Baker’s yeast

Brewer’s yeast

3. Receptor activation…

Receptor Specificity:

Yeast has 2 distinct GPCR signalling cascades

Versele et al. 2001

3. Receptor activation…

Signalling Specificity is achieved by Scaffolding in Yeast

Cartoon of Ste5p and Far1p scaffolds. Ste5p is required for activation of the mating MAPK cascade in response to mating pheromone and does not have an intrinsic kinase activity. Far1p is required for oriented polarized growth in response to mating pheromone. Far1p is postulated to be an analog of Ste5p on the basis of its ability to associate with multiple components of an individual signal transduction pathway, but it is not known whether they simultaneously bind to associated signaling components.

3. Receptor activation…

In mammalian cells,

GPCR specificity is illustrated by GPCR mediated activation of MAPK cascades

7TM receptors activate the ERK/MAPK cascade by several different pathways

Nature Reviews Molecular Cell Biology3; 639-650 (2002)

3. Receptor activation…

Scaffolding of MAPK cascade is also seen in mammalian cells

Nature Reviews Molecular Cell Biology3; 639-650 (2002)

3. Receptor activation…

Microdomains can also contribute to GPCR specificity: Caveolae

Schematic representation of the lipid and protein organization of a caveola. Sphingolipid- and cholesterol-rich domain is shown in red and nonraft lipid domains are shown in blue. Caveolae contain a coat of oligomeric caveolin molecules inserted into the cytoplasmic leaflet of the membrane. Some proteins, including certain GPCR (shown as heptahelical structures with associated G protein), partition to caveolar domains due to either acylation, binding to caveolin or formation of a sphingolipid ‘shell’ around the protein (or by a combination of these, and/or yet unknown, mechanisms). Also shown are undefined cytoskeletal interacting proteins (orange, green, purple) and noncaveolar membrane proteins (blue) and partners (light blue).

3. Receptor activation…

Diversity: multiple GPCRs are expressed in the same cell/tissue

Example of blood vessels- molecular biology is trying to understand the

complexity of GPCR responses seen in in vivo situations.

3. Receptor activation…


Example of cardiac cells.

British Journal of Anaesthesia, 2004, 93;34-52

Sympathetic and parasympathetic signalling cascades of G-protein coupled receptors down to the level of cellular responses. Note the intimate crosstalk between the various signalling pathways. Lines with blunted ends (=) indicate inhibition. AC=adenylyl cyclase; ACh=acetylcholine; AR=adrenergic receptor; cAMP=cyclic AMP; cGMP=cyclic GMP; DAG=diacylglycerol; ET1=endothelin receptor-1; GC=guanylyl cyclase; G  i, G  s, G  q, Gß  =G-protein subunits; IP3=inositol trisphosphate; M2=muscarinic acetylcholine receptor; MAPK=mitogen activated protein kinase; NOS=nitric oxide synthase; PDK1=phosphoinositide-dependent kinase-1; PI3K=phosphoinositide-3 kinase; PKA, PKB, PKC, PKG=target-specific serine–threonine protein kinases; PLC=phospholipase C; Ras=small monomeric GTPase; RNOS=reactive nitric oxide species.