Multistate receptors and multiple ligand binding sites

In the multistate model (Schwartz et al., 1995), the receptor is proposed to alternate spontaneously between multiple active and inactive conformations. The key elements in this model are:

• The biological response to a given ligand is determined by the receptor conformation to which this ligand binds with highest affinity. If the preferred conformation is recognized by the G protein as active, the ligand will behave like an agonist. If the preferred conformation is inactive, the ligand will behave like an inverse agonist.

• Two agonists acting at the same receptor do not necessarily have to share an overlapping binding site; they both must stabilize an active conformation.

• An overlap in the binding site of the agonist and a competitive antagonist is not required. The agonist and antagonist simply stabilize distinct receptor conformations to which they bind in a mutually exclusive fashion.

This model implies that there are multiple ways of propagating activation of GPCRs or, in other words, there is no common 'lock' for all agonist 'keys'. Support for the existence of non-overlapping binding sites is provided by a number of observations:

• In contrast to the rhodopsin-like family A receptors, the family C metabotropic glutamate receptors contain two domains that act synergistically to produce receptor activation: a very large extracellular N-terminal agonist binding domain and the 7-TM helices involved in receptor activation and G protein coupling. Interestingly, mutagenesis experiments have identified crucial amino acids required for binding of (allosteric) antagonists in TM3, TM6 and TM7 of these receptors, whereas no extracellular N-terminal regions appear necessary (Figure 210).

• Adrenergic, muscarinic and AT1 receptor-activating antibodies are present in serum from patients with different pathologies (Table 24). These antibodies are directed against the extracellular loop regions of these receptors; antibodies directed against synthetic peptides mimicking such loop regions have also

Figure 210 Proposed secondary structure of metabotropic glutamate receptor homodimers. Glutamate is bound between two globular lobes in the N-terminal extracellular region whereas allosteric modulators (AM) bind at TM3 (not shown) TM6 and TM7. Reprinted from Trends in Pharmacological Science, 24, Pellegrini-Giampietro, D. E., The distinct role of mGlul receptors in post-ischemic neuronal death, 461-470. Copyright (2003), with permission from Elsevier.

Figure 210 Proposed secondary structure of metabotropic glutamate receptor homodimers. Glutamate is bound between two globular lobes in the N-terminal extracellular region whereas allosteric modulators (AM) bind at TM3 (not shown) TM6 and TM7. Reprinted from Trends in Pharmacological Science, 24, Pellegrini-Giampietro, D. E., The distinct role of mGlul receptors in post-ischemic neuronal death, 461-470. Copyright (2003), with permission from Elsevier.

Table 24 Examples of functional GPCR antibodies.

Receptor Disease Effect of AAB. Epitope localization

Table 24 Examples of functional GPCR antibodies.

Receptor Disease Effect of AAB. Epitope localization

arR

hypertension

agonist-like

loop 1, 2

ßi-R

dilated cardiomyopathy

agonist-like

loop 1,2

ßi-R

myocarditis

agonist-like

loop 1,2

ßi-R

Chagas' disease

agonist-like

loop 2

ß2-R

Chagas' disease

agonist-like

loop 2

ß2-R

allergic asthma

inhibitory

loop 3

AT1-R

preeclampsia

agonist-like

loop 2

AT1-R

malignant hypertension

agonist-like

loop 2

AT1-R

vascular renal rejection

agonist-like

loop 2

muscarinic M2-R

Chagas' disease

agonist-like

loop 2

muscarinic M2-R

dilated cardiomyopathy

agonist-like

loop 2

5HT4-R

systemic lupus erythematosus

agonist-like

loop 2*

been shown to display agonistic properties (Figure 211). This implies that some GPCRs can be activated by the penetration of their small natural agonist into the central cleft as well as by the interaction of bulky antibodies with extracellular loop regions. It is still not clear whether the activated receptor displays the same conformation in both cases and, in fact, little is still known about the molecular e 40

e 40

Antibody log(M)

Figure 211 Dose-response curve of affinity-purified pa-adrenergic receptor autoantibodies from patients with dilated cardiomyopathy. Reprinted from Journal of Molecular and Cellular Cardiology, 27, Wallukat, G., Wollenberger, A., Morwinski, R., Pitschner, H. F., Anti-betal-adrenoceptor antibodies with chronotropic activity from the serum of patients with dilatated cardiomyopathy: localization of two epitopes in the first and second extracellular loops, 397-406. Copyright (1995) with permission from Elsevier.

Extracellular «

Extracellular «

Figure 212 Structure of the M2 muscarinic receptor with indicating the natural 'orthosteric' ligand-binding site and the binding site for allosteric modulators. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Drug Discovery, 1, Christopoulos, A., Allosteric binding sites on cell-surface receptors: novel targets for drug discovery, 198-210, © (2002).

Figure 212 Structure of the M2 muscarinic receptor with indicating the natural 'orthosteric' ligand-binding site and the binding site for allosteric modulators. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Drug Discovery, 1, Christopoulos, A., Allosteric binding sites on cell-surface receptors: novel targets for drug discovery, 198-210, © (2002).

mechanism of antibody activation in general. As a typical example, it was initially assumed that antibodies against the second extracellular loop of the p2-adrenergic receptor could stabilize the receptor in its 'active' conformation. It is now thought that they act by dimerizing the receptor and that the conformation they induce is different from the (monomeric) resting state and the fully active conformation that is induced by small agonist molecules (Mijares et al., 2000).

• Even though the majority of ligands seem to bind deep within the central cleft of biogenic amine receptors, mutation studies reveal that certain small antagonist molecules may partly interact with residues closer to the surface of the membrane (Figure 212). This has been most extensively studied for muscarinic and adrener-gic receptors. As may be expected, such antagonists show no structural relationship with the natural agonists of these receptors.

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