Activated GPCRs interaction with G proteins

Recently, X-ray crystallography has provided substantial insight into the tertiary structure of the heterotrimeric G proteins. These studies reveal that the a subunits consist of a ras-like GTPase domain (six-stranded P sheets surrounded by six a helices) and a helical domain. The bound guanine nucleotide is deeply buried between these two domains. Whereas a and P subunits make extensive contacts, there appear to be no direct interactions between a and y subunits (Figure 110).

The three-dimensional structure of a light-activated state of rhodopsin was recently obtained and modelling studies reveal that transducin must alter its conformation to bind to the activated receptor. Rather than a 'lock' and 'key' fit, interactions between receptors and G proteins should include an 'induced fit' (analogous to that proposed by Koshland to describe enzyme-substrate interactions) at the heart of the signal transduction mechanism (Yeagle and Albert, 2003).

Pending the resolution of the atomic structure of a receptor-G protein complex, still little is known about the actual points of interactions between the receptor and the G protein and, thus, how the two proteins are oriented relative to one another. This would help us to understand how hundreds of different GPCRs with remarkably diverse amino acid sequences can all interact with a similar subset of G proteins. Presently, the model for GPCR-G protein interaction includes the following elements:

• GPCR activation enables the a subunit of a G protein (further denoted as Ga subunit) to interact with previously inaccessible receptor residues located within the endo2 and endo3 loops and the C-terminal tail.

• p—y complexes increase the affinity of Ga for activated GPCRs but it is not clear whether the p—y complexes also make specific contacts with the receptor proteins.

• GPCR binding induces changes in the Ga conformation. This will lead to a weakening of GDP binding and eventually GDP release. Because it is unlikely that the nucleotide can be contacted directly by the receptor protein (Figure 110), GPCRs are thought to trigger GDP release by an allosteric mechanism.

When activated by appropriate agonists, most GPCRs preferentially activate a limited set of G proteins (Table 10). Mutation studies show that multiple intracellular receptor regions act in a co-operative fashion to get correct G protein recognition and efficient G protein activation (Table 11). They are (Wess, 1998):

• The Asp/Glu-Arg-Tyr triplet at the N-terminal region of endo 2.

• Residues in the N- and C-terminal regions of endo 3.

• A few residues in the N-terminal region of the cytoplasmic C-terminal tail (including the highly conserved NPXXY sequence of family A GPCRs), but deletion mutagenesis studies have shown that most of the tail itself is not required for efficient G protein coupling.

Differences in G protein recognition pattern among two receptors can be attributed to subtle differences in those intracellular regions as well as in their co-operative behavior. Hence, the selectivity of G protein recognition may vary among different classes of GPCRs and even among structurally closely related members of the same receptor subfamily (adrenergic receptors represent a nice example of this) (Figure 111).

A large body of evidence indicates that certain amino acid residues in the C-terminal portions of Ga can directly contact the receptor protein and play a key role in dictating the specificity of coupling (Table 12). Since this region is fairly similar for Ga components of the same G protein family, receptors will often be capable of stimulating all the members of this family. Hence, based on their G protein-coupling preference, GPCRs can be broadly subclassified into Gi/o-, Gs- and Gq/11-coupled receptors. In this respect, it is of notice that replacing the last five amino acids of Gaq with the corresponding Ga; or Gao sequences allows traditional Gi/o-coupled receptors to stimulate PLC-P (a typical Gq/11-mediated response).

Figure 110 Proposed binding of a G protein heterotrimer to an activated GPCR. Principles of Biochemistry by Albert L. Lehninger, et al. © 2000, 1993, 1982 by Worth Publishers. Used with permission.

Studies performed with recombinant cell systems have clearly established that, although most GPCRs are preferentially coupled to a certain subfamily of G proteins, they can also activate other classes of G proteins, but with reduced efficiency. The first evidence for such multiplicity of G protein coupling arose from studies indicating that, in addition to inhibiting adenylate cyclase, some Gi/o-coupled receptors mediate inositol phosphate production through a pertussis toxin-insensitive pathway. In some cases, a single receptor was found to simultaneously activate members of three or even four unrelated classes of G proteins (Table 13). Many factors may affect the recep-tor-G protein interaction (Figure 112).

In general, the nature of the observed response not only depends on which G protein is preferentially recognized by the receptor, but also by which ones are present in the studied cell or tissue. If more than one type of G protein is activated in a single cell,

Table 10 Preferred GPCR-G protein interactions. Reprinted from Pharmacology and Therapeutics, 80, Wess, J., Molecular basis of receptor/G protein-coupling selectivity, 231-264. Copyright (1998), with permission from Elsevier.

Peptides Biogenic amines

Table 10 Preferred GPCR-G protein interactions. Reprinted from Pharmacology and Therapeutics, 80, Wess, J., Molecular basis of receptor/G protein-coupling selectivity, 231-264. Copyright (1998), with permission from Elsevier.

Peptides Biogenic amines

Angiotensin II (AT1a, b)

Gq/11

Muscarinic receptors: m1, m3,

Gq/11

m5)

Angiotensin II (AT2)

Gi/o

muscarinic receptors: (m2, m4)

Gi/o

Bombesin (BB1-3)

Gq/11

Dopamine (D1, D5)

Gs

Bradykinin (Bb B2)

Gq/11

Dopamine (D2, D3, D4)

Gi/o

C5a Anaphylatoxin

Gq/11

adrenoceptors: (a 1a, a1b, a1d)

Gq/11

Cannabinoids (CB1, CB2)

Gi/o

adrenoceptors: (a2a, a2b, a2c)

Gi/o

Chemokines (CCR1-CCR5)

Gi/o

adrenoceptors: (ß1, ß2, ß3)

Gs

CCK/Gastrin (CCKA, CCKB)

Gq/11

Histamine: H1

Gq/11

ET (ETa, ETb)

Gq/11

Histamine: H2

Gs

Galanin

Gi/o

5-HT (5-HT1a-f)

Gi/o

Gonadotropin-releasing horm.

Gq/11

5-HT (5-HT2a-c)

Gq/11

Melatonin (MEL1A, 1B)

Gi/o

5-HT (5-HT4, 6, 7)

Gs

Melanocortins (MC1-5)

Gs

Neuropeptide Y (Y1-5)

Gi/o

Neurotensin

Gq/11

Oxytocin

Gq/11

Opioid peptides (|l, K, 8)

Gi/o

Somatostatin (SSTR1-5)

Gi/o

Tachykinins (NK1-3)

Gq/11

Thyrotropin-releasing hormone

Gq/11

Vasopressin (V1a, V1b)

Gq/11

Vasopressin (V2)

Gs

the magnitude of the different responses (and therefore the ratio of their magnitude) will be affected by the concentrations of these G proteins, their effectors, and the further downstream signalling components. In this respect, multiplicity in G protein coupling is frequently observed in artificial expression systems. However, because

Table 11 Mutagenesis of a2-adrenergic receptors differently affect adenylate cyclase (AC) stimulation (via Gs) and inhibition (via Gj/o). (I = intracellular loop).

Region

Mutation

AC inhibition

AC stimulation

13 N-terminus I3 C-terminus

substitution deletion substitution basic residues

no effect

+

no effect

Figure 111 G protein-coupling preference of a-j-, a2- and p-adrenergic receptors.

both receptors and G proteins are usually expressed at rather high levels, it has been argued that receptor/G protein combinations in these co-expression experiments are not necessarily of physiological relevance. This raises the question of whether such complex signalling reveals artefactual promiscuous coupling or whether it is a genuine property of GPCRs.

Table 12 C-terminal portions of Ga subunits. Reprinted from Pharmacology and Therapeutics, 80, Wess, J., Molecular basis of receptor/G protein-coupling selectivity, 231-264. Copyright (1998), with permission from Elsevier.

- 7

- 6

- 5

- 4

- 3

- 2

- 1

as

L

R

Q

Y

E

L

L

aolf

-

L

K

Q

Y

E

L

L

aq, 11

-

L

K

E

Y

N

L

V

a14

-

L

R

E

F

N

L

V

a15, 16

-

L

D

E

I

N

L

L

Oil, 2

-

L

K

D

C

G

L

F

Oi3

-

L

K

E

C

G

L

Y

ao1, 2

-

L

R

G

C

G

L

Y

at1, 2

-

L

K

D

C

G

L

F

az

-

L

K

Y

I

G

L

C

agust

-

L

K

D

C

G

L

F

«12

-

L

K

D

I

M

L

Q

«13

-

L

K

Q

L

M

L

Q

Table 13 Typical examples of receptors showing multiplicity in G protein coupling. 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.

Receptor

Gs

Gi

Gq/11

G12

Adenosine (A3)

X

X

(a2-Adrenergic

X

X

ß-Adrenergic

X

X

Corticotropin- releasing hormone

X

X

X

Dopamine (Dj)

X

X

Metabotropic glutamate (la)

X

X

X

Endothelin (ETB)

X

X

Galanin

X

X

X

Glucagon

X

X

Gonadotrophin releasing hormone

X

X

X

Histamine H2

X

X

Luteinizing hormone

X

X

X

Melatonin

X

X

Muscarinic (ml and m3)

X

X

Neurotensin

X

X

Pancreastin

X

X

Parathyroid hormone

X

X

Platelet-activating factor

X

X

Prostacyclin

X

X

X

Prostaglandin (EP3D)

X

X

Serotonin (5-HT2C)

X

X

Sphingosine 1-phosphate (Edg3)

X

X

X

Substance P

X

X

Thyrotropin

X

X

X

X

Thrombin

X

X

X

Vasopressin VJa

X

X

Vasoactive intestinal peptide

X

X

Considerations in favour of artefactual coupling:

• Whereas several tachykinins are able to stimulate cAMP as well as IP3 production in CHO cells expressing a recombinant NK-1 receptor, only the latter response is observed in cells that express the endogenous receptor (Torrens et al., 2000).

• Distinct coupling properties of the thyrotropin receptor have been observed when comparing intact cells with cell membranes (Allgeier et al., 1997) and this suggests that disruption of the cellular organisation may favour the promiscuous coupling of GPCRs. In this respect, it is worth mentioning that the signalling specificity at the G protein level is often studied on cell homogenates or membrane preparations.

Figure 112 The coupling of GPCRs with multiple G proteins is selectively regulated at different levels. 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.

Figure 112 The coupling of GPCRs with multiple G proteins is selectively regulated at different levels. 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.

Considerations in favour of a genuine property:

• Some GPCRs also interact with multiple G proteins in non-transfected cells. For example, activation of the thyrotropin receptor in dog thyroid membranes led to increased incorporation of [a32P]GTP azidoanilide into Gas, Gaq and Ga;. In addition, pretreatment of intact thyrocytes with pertussis toxin allows TSH to shift from (Gi/o-mediated) adenylate cyclase inhibition to (Gs-mediated) adenylate cyclase stimulation.

• Similarly, endogenousely expressed a2B-adrenergic receptors have been found to interact with Gaq in addition to Ga in neuroblastoma x glioma cells (Holmberg et al., 1998).

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