REVIEWS TIBS 23 – NOVEMBER 1998 418 0968 – 0004/98/$ – See front matter © 1998, Elsevier Science. All rights reserved. PII: S0968-0004(98)01287-0 THE LARGE MULTIGENE family of G- protein-coupled receptors (GPCRs) plays a fundamental role in cellular func- tion. These proteins evolved early and can be found in a variety of organisms ranging from yeast and Dictyostelium to mammals. Several hundreds of distinct GPCRs couple extracellular signals as di- verse as photons, odors, monoamines, peptides and ions to distinct intracellu- lar signals and are estimated to be the target for around 60% of drugs currently available. For several years, it has been accepted that, upon activation by ag- onists, GPCRs transfer the signal to the family of heterotrimeric G proteins, which, in turn, can either stimulate or inhibit the activity of a wide variety of cellular effector proteins through the release of  and  subunits 1 (Fig. 1). Exogenous interference with these ver- satile signaling systems by (partial) ag- onists (to stimulate the system) or antag- onists (to prevent action of endogenous agonist) are central to current pharma- cotherapy for a wide variety of human diseases. Agonist-independent GPCR activity Recently, our concepts of GPCRs have been firmly challenged. It is now well es- tablished that GPCRs can signal without agonist stimulation. Since the cloning of the gene encoding the hamster  2 adren- ergic receptor in 1986 (Ref. 2), many re- lated genes encoding GPCRs have been cloned (see the GPCRDB database at http://swift.embl-heidelberg.de/7tm/). The availability of this genetic infor- mation allowed detailed analysis of (mu- tant) GPCRs in heterologous expression systems. Spontaneous or constitutive GPCR activity was convincingly de- scribed in 1989 for the -opioid receptor, endogenously expressed in NG108-15 neuroblastoma-glioma cells 3 , but was only fully appreciated after the reports of constitutive activity of overex- pressed, mutant  1B and  2 receptors 4 . Constitutive, agonist-independent sig- naling has now been observed for a wide variety of GPCRs, in various sys- tems 5,6 , including transgenic mice 7 . Various GPCRs ligands, formerly known as antagonists (see Box 1) but now de- scribed as inverse agonists, have been shown to inhibit constitutive GPCR sig- naling. However, not all antagonists af- fect GPCR activity; neutral antagonists do not alter basal signaling but are able to prevent the action of both agonists and inverse agonists by occupying the GPCR binding site 5,6,8 . An ongoing discussion concerns the underlying receptor theory of constitutive GPCR activity and inverse agonism 5,6,8–11 . Experimental evidence obtained with several GPCRs indicates that structural constraints keep the GPCR in an inactive (R) conformation that prevents an effec- tive interaction between peptide se- quences in the intracellular loops of the GPCR and the G protein 12 . Intrahelical salt bridges between transmembrane domains 3 and 7 have, for example, been proposed to constrain the conformation of rhodopsin 13 and the  1B receptor 14 . Agonist binding or mutations in so- called constitutively active mutant (CAM) GPCRs are believed to relieve the constraints and to cause the GPCR to adopt an active (R*) conformation. R. Leurs, A. E. Alewijnse and H. Timmerman are in the Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands; and M. J. Smit is at the Mount Sinai School of Medicine, Dept of Pharmacology, New York, NY 10029, USA. Email: leurs@chem.vu.nl Agonist-independent regulation of constitutively active G-protein-coupled receptors Rob Leurs, Martine J. Smit, Astrid E. Alewijnse and Henk Timmerman G-protein-coupled receptors constitute one of the largest protein super- families in mammals. Since the cloning of the encoding genes, these im- portant drug targets have been subjected to thorough biochemical and pharmacological studies. It has become clear that G-protein-coupled re- ceptors not only transmit signals after stimulation by agonists but can also spontaneously couple to signal-transduction pathways. Recent findings show that constitutively active G-protein-coupled receptors can also be regulated in an agonist-independent manner, which has important impli- cations for the interpretation of the actions of (inverse) agonists and the results of site-directed-mutagenesis studies. COOH NH2 Effector g b a Cellular response + Figure 1 A G-protein-coupled receptor (GPCR) and its signaling pathways. GPCRs can couple to a variety of heterotrimeric G proteins that are assembled from  (one of 16),  (one of five) and  (one of seven) subunits. After GPCR coupling the G protein dissociates into  and  subunits. The  and the  subunits can modulate the activity of a variety of effector proteins – for example, adenylyl cyclase, phospholipase A 2 , phospholipase C or ion channels.