Constitutive Receptor Activity series
Historical review: Negative efficacy and the constitutive activity of G-protein-coupled receptors

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The idea that a receptor can produce signalling without agonist intervention and that several antagonists can be ‘active’ in repressing such spontaneous activity is contained in the concept of ligand-induced conformational changes. Yet, this idea was neglected by pharmacologists for many years. In this article, we review the events that brought inverse agonism and constitutive activity to general attention and made this phenomenon a topic of current research. We also suggest a classification of antagonists based on the cooperativity that links their primary site of interaction with other functional domains of the receptor.

Section snippets

Negative efficacy

Receptor science is a charming example of how concepts go hand in hand with experiments in biology. New concepts are introduced to understand phenomena, tame their complexity and find the hidden general meaning. Yet, are the experiments that bring life to concepts, by repeatedly proving their strength and broadness, sometimes declaring their death? The receptor itself was born as a concept and remained an idea for a long time. It took at least 70 years before advances in single-channel

Allosteric modulators of GABAA receptor channels and negative antagonists of 7TM receptors

It is ironic that the compounds that gave birth to the term inverse agonist, β-carbolines and many other compounds of diverse structural classes, are not true inverse agonists in a strict sense. The first β-carboline was extracted from human urine and brain tissue in an attempt to identify an endogenous ligand for the ‘benzodiazepine receptor’ [4]. GABA, known to enhance benzodiazepine binding, had the opposite effect on β-carboline affinity [5]. As shown later, β-carbolines elicited an

Ligand efficacy is the result of cooperativity in biological macromolecules

As mentioned earlier, negative efficacy was not a new concept. It is, in fact, a direct manifestation of the inherent property of any protein: the presence of cooperative interactions among its functional domains. External and internal interactions in proteins are driven by the same kind of forces, and thus no perturbation can affect one type of interaction without also changing the other type of interaction [9].

The first and most-elegant analysis of cooperativity was performed by Wyman, with

Experimental evidence of negative efficacy

The first evidence of negative efficacy was obtained in the test-tubes of a reconstituted signaling system. Using purified β2-adrenoceptors and Gs proteins reinserted into lipid vesicles, Cerione et al. [24] demonstrated that ‘empty’ receptors are active and can be stereoselectively inhibited by an antagonist. Although this evidence was criticized and ‘blamed’ on the ability of thiols (a component of the assay mix) to activate the receptor [25], the same indication reappeared a few years later

Constitutive activity is a frequent outcome of GPCR mutations

The turning point that sparked the surge of general interest in negative efficacy was the discovery, first made in the α1B-adrenoceptor, that certain mutations in the sequence of GPCRs can cause a dramatic increase in the intrinsic constitutive activity of the receptor [31]. Antagonists prevented this effect, suggesting that the mutations had lowered the intrinsic ‘set-on’ point for constitutive activation. A follow-up study, in which the single residue A293 was systematically replaced with all

Stretching the ternary complex model

A detailed study of the biochemical properties of a CAM β2-adrenoceptor phenotype disclosed a peculiar ‘signature’ of what turned out to be a constitutively active receptor [33]. The apparent affinities of β2-adrenoceptor agonists for this CAM receptor were enhanced in a manner related to their intrinsic efficacy at the wild-type receptor: the greater the signalling strength of the agonist, the larger the boost in affinity produced by the mutation. Yet, the biphasic shape of the agonist binding

The past 10 years of constitutive activity

Following these initial studies, a wave of investigations of constitutive activity and inverse agonism propagated to virtually any known GPCR. The past 10 years have seen an exponential growth of published articles on such topics, as covered in many reviews 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 but they also mark the end of our historical perspective. We shall now briefly discuss some issues that remain unresolved in this field.

Concluding remarks

Spontaneous activity and negative efficacy of receptors, once considered an oddity, have provided a new dimension to the study of the functional chemistry of receptors. Novel crucial questions have emerged. Answering such questions is important because it will provide insight into the fundamental mechanisms of receptor function and provide new strategies to design chemicals that can control the subtleties of the molecular behaviour of receptors. Again, the alternating dance between concepts and

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