Receptor activation

The basal activity of a wild-type GPCR might vary from totally inactive (AT1a receptor) to partially active, depending on the nature of the GPCR. Partially active receptors are referred to as 'constitutive active receptors' (constitutive activity being defined as agonist-independent activity). The p2-adrenergic receptor is one of them; in transfected cell systems it triggers second messenger (cAMP) production in the absence of agonist.

The greatest insights into the molecular basis of GPCR activation have come from the analysis of mutant receptors. However, there are many issues to consider regarding its interpretation. Most useful are single or multiple point mutants. Such mutagenesis is generally characterized as either loss-of-function (i.e. decrease in binding affinity or functional response) mutants or gain-of-function mutants. All loss-of-function mutants are subject to problems of interpretation as they may result from the loss of critical interactions between receptor and agonist, from a mis-folded receptor structure or reduced receptor expression levels. Gain-of-function mutagenesis generally introduces or re-introduces binding and/or function of an 'inactive' receptor.

The functions of residues are most clearly categorized by alanine substitution mutations. These delete the side chain of the amino acid beyond the P-carbon,

Figure 102 Filler residue mutations produce a null effect. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 102 Filler residue mutations produce a null effect. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

leaving a small 'hole' in the receptor structure. With regard to receptor activation, such point-substitution mutation may have four basic outcomes (Figures 102 to 105):

• It may produce a null effect (Figure 102). Residues that tolerate multiple substitutions can be regarded as plugging functionally unimportant gaps in the receptor structure (i.e. filler residues).

• It may induce a simple reduction of the structural stability of the receptor (Figure 103). The mutation of such a stabilizer residue may reduce the expression level of the receptor by lessening its probability of folding successfully, and undergoing correct trafficking. However, it should not affect the signalling ability of those receptor molecules because it does not impair receptor activation.

• A mutation may increase the basal activity of the receptor (Figure 104). Such constraining residues are likely to contribute to maintain the receptor in its inactive state by forming intramolecular bonds. As for the mutant, these bonds are weakened or broken in the ligand-activated state of the receptor.

Figure 103 Stabilizer residue mutations tend to decrease receptor expression. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 103 Stabilizer residue mutations tend to decrease receptor expression. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 104 Constraining residue mutations produce constitutive receptor activity. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., AHman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 104 Constraining residue mutations produce constitutive receptor activity. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., AHman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

• The mutation may reduce the signalling efficacy of the receptor (Figure 105). This indicates that the target residue makes interactions necessary for the activated conformation of the receptor. The interactions made by such an activator residue may either be intramolecular, or intermolecular (i.e. with the agonist or G protein).

There are major differences in the molecular mechanisms of activation between GPCRs: some receptors are easy to activate and many different single mutations activate them (adrenergic or TSH receptors), when others (AT1 receptor) require more complex molecular changes. In general, it is believed that:

• In the ground state, the receptors are constrained in an inactive conformation by a network of intramolecular constraining interactions; and

Figure 105 Activator residue mutations decrease signalling efficacy. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 105 Activator residue mutations decrease signalling efficacy. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

• The presence of an agonist is responsible for the release of these inactivating constraints and the creation of a new network of activating interactions resulting in the formation and/or stabilization of the active state of the receptor.

• This activation process possibly occurs in a multistep sequence. In this respect, it has been proposed that small molecules should mainly act by destabilizing constraining interactions and that peptide ligands should mainly act by creating 'activator contacts'.

Compelling evidence for the existence of constraining intramolecular interactions that normally keep the ligand-free receptor inactive was obtained by mutation experiments involving the substitution of single amino acids. It was observed that certain substitutions might produce receptors that have higher basal activity as compared to the wild-type counterparts (these are termed 'constitutive active receptor mutants' or 'CAMs') (Figure 106). A dramatic example of this was provided by mutation of Ala293 (Ala6 34)

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Figure 106 Basal inositol phosphate (IP) production in recombinant cells with wild-type (wt) AT1A receptors and L305Q constitutively active mutant (CAM) receptors (production increases with receptor expression). Reprinted from Proceedings of the National Acadademy of Science USA, 97, Parnot, C., Bardin, S., Miserey-Lenkei, S., Guedin, D., Corvol, P. and Clauser, E., Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay, 76157620. Copyright (2000) National Academy of Sciences, USA.

in the C-terminal part of the third intracellular loop of the alB adrenergic receptor: any substitution by one of the 19 other amino acids resulted in full receptor activation (Kjelsberg et al., 1992). This suggests that Ala293 is implicated in an intramolecular interaction (with Glu289 on TM6) that prevents the receptor from being active in the absence of agonist. The marked structural instability and enhanced conformational flexibility of constitutively activated ^-adrenergic and histamine H2-receptor mutants (compared to the wild-type receptors) add further support to the existence of stabilizing/ constraining intramolecular interactions when a wild-type receptor is at rest.

Comparison of several CAMs in family A GPCRs suggests that the conserved D/ERY motif (at the cytoplasmic end of TM3) plays a major role in constraining their inactive conformation. Charge-neutralizing mutations, which mimic the protonated state of the aspartic acid/glutamic acid in this motif, cause dramatic constitutive activation of, for example, the a1B- and the p2-adrenergic receptors. It is therefore believed (i.e. the 'protonation hypothesis') that protonation of the aspartic acid/glutamic acid in this motif is at least one of the key events in the activation of this GPCR family.

Additional conformational constraints may also be operative for certain GPCRs (Figure 107). For example, random mutagenesis of the ATlA receptor revealed that the substitution of several amino acids present on its TM regions are prone to produce CAMs and it is of notice that several of those amino acids are located on one side of TM3. Taken together, such mutation studies support the notion that TM3 and TM6 play a very general role in the conformational changes associated with GPCR activation. However, it should be mentioned that individual mutations that cause constitutive activation of one receptor type do not always do so when transferred to another. This indicates that the primary interhelical contacts have been tailored to suit the properties of individual receptors. Most CAMs are supposed to release the conformational constraints of the GPCR inactive state without creating new interactions. Such mutants help our understanding of the structure of the inactive state, but not about the structure of the ligand-induced active conformation.

The conformations of these CAMs are thus approximations of the real active conformation and this represents a severe limitation for the study of GPCR activation. In this respect, mutations mimicking activator interactions have been suggested to be better models of ligand-activated receptor conformations.

The occurrence of activator interactions is supported by the finding that certain GPCRs, like the ATj receptor, are difficult to activate constitutively by a single mutation. Most mutations only double the basal activity of the receptor, which is far below the maximal agonist stimulation. One possible explanation is that several constraining interactions need to be overcome to get full receptor activation. Another explanation is that the binding of an agonist generates a set of activator interactions, resulting in additional changes in the receptor conformation.

Still very little is known about the structural changes that go along with GPCR activation. The three-dimensional structure of a light-activated state of rhodopsin (metarhodopsin II) was recently obtained by X-ray crystallographic studies. However, it should be remembered that rhodopsin is unique among the GPCRs in that its ligand is covalently bound to the receptor and that, upon absorption of a photon, it isomerizes

Figure 107 Amino acids (in black) whose mutations lead to strong constitutive activity in four representative GPCRs. (a) Human p2-adrenergic receptor, (b) rat AT1A receptor, (c) yeast a-factor receptor, (d) human TSH receptor. Reprinted from Trends in Endocrinology and Metabolism, 13, Parnot, C., Miserey-Lenkei, S., Bardin, S., Corvol, P. and Clauser, E., Lessons from constitutively active mutants of G protein-coupled receptors, 336-343. Copyright (2002), with permission from Elsevier.

Figure 107 Amino acids (in black) whose mutations lead to strong constitutive activity in four representative GPCRs. (a) Human p2-adrenergic receptor, (b) rat AT1A receptor, (c) yeast a-factor receptor, (d) human TSH receptor. Reprinted from Trends in Endocrinology and Metabolism, 13, Parnot, C., Miserey-Lenkei, S., Bardin, S., Corvol, P. and Clauser, E., Lessons from constitutively active mutants of G protein-coupled receptors, 336-343. Copyright (2002), with permission from Elsevier.

to an agonist within the binding pocket. Thus, the process of ligand binding is not an integrated part of the activation process. The three-dimensional structural determination of typical agonist-bound GPCRs has not yet been determined by X-ray crystallography, but mutation studies and biochemical and biophysical approaches (especially with P2-adrenergic receptors and rhodopsin) have provided indirect information about the structural changes that go along with family A GPCR activation. Biochemical and spectroscopic approaches to study receptor activation include:

• The generation of artificial 'bridges' between two TM helices. These will prevent the separation and rotation of the involved TM domains and, hence, may prevent receptor activation, at least if such structural changes are required. In this respect, several cysteine-cysteine disulfide bridges have also been shown to prevent light activation of rhodopsin.

Extracel side

Extracel side receptor activation

receptor activation

O Fluorescence in lipid ^ fluorescence in potar environment

Figure 108 Orientation of the environment-sensitive fluorophores 125Cys-NBD and 285Cys-NBD at the extracellular side of the p2-adrenergic receptor (top view). Left: inactive receptor, Right: receptor with rotated TM3 and TM6 in active conformation (according to Gether, 1998).

• Zinc-binding sites can be formed by introducing pairs of histidines in positions predicted to be in close proximity. Zinc binding to such engineered sites provides information about the proximity of the histidine residues and, hence, about the orientation of the involved TM domains. In this respect, a bridge joining TM3 and TM6 was found to prevent the activation of, for example, NK-1 receptors and rhodopsin.

• Spectroscopic approaches with wild-type or mutant receptors having single or a limited number of cysteines. This provides information about whether the environment of a side-chain is aqueous or hydrophobic and whether it is buried. They include:

- Electron paramagnetic resonance spectroscopy with sulfhydryl-specific nitroxide spin labels (whose unpaired electrons can be probed spectroscopi-cally). Such spin labels can indicate whether the environment of a side-chain is aqueous or hydrophobic and whether it is buried.

- Covalent labelling of receptors with cysteine-reactive fluorescent probes whose fluorescence is highly sensitive to the polarity of their environment (Figure 108).

The current picture is as follows: TM3 is very highly tilted and longer than the other TM helices. Thus, TM3 makes multiple interhelical contacts and its movement is therefore ideally placed to propagate conformational changes induced by agonist binding through the transmembrane structure of the receptor. Studies with rhodopsin, P2-adrenergic and muscarinic receptors suggest that TM3 and TM6 play critical roles for the transition of family A GPCRs to their fully activated state. This should involve a counter-clockwise rotation of these TM domains and an outward (away from the bundle) movement of their cytoplasmic ends (Figure 109). Other helices probably also adjust their positions upon activation. As a result, the helical bundle is thought to blossom open at its cytoplasmic end. This enables the G proteins to interact with previously inaccessible receptor residues located within the endo2 and endo3 loops and the C-terminal tail.

Figure 109 Model for muscarinic receptor activation. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Figure 109 Model for muscarinic receptor activation. Reprinted from European Journal of Pharmacology, 375, Hulme, E. C., Lu, Z. L., Ward, S. D., Allman, K. and Curtis, C. A., The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm, 247-260. Copyright (1999), with permission from Elsevier.

Based on the model of rhodopsin (family A GPCR), the three-dimensional structure of the GLP1 receptor (family B GPCR) in the absence or presence of its endogenous hormone GLP1 has recently been proposed. These molecular modelling studies suggest that both receptors may undergo the same kind of structural changes upon activation.

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