Introduction

The majority of hormones, neurotransmitters, and other chemical signalling molecules in the human body exert their effects via G protein coupled receptors (GPCRs) (Strader et al. 1994; Ji et al. 1998; Gether 2000). Evidently, this extreme functional diversity raises some fundamental questions in relation to the function of GPCR at the molecular and cellular level. What are, for example, the molecular mechanisms underlying the ability of this broad variety of molecules to bind and activate receptors that presumably share a preserved overall tertiary structure? Or more specifically, what are the physical changes linking binding of an agonist to activation of intracellular signalling cascades? During the last few years our insight into these fundamental mechanisms have improved considerably. An important breakthrough has been the application of biophysical approaches that has allowed direct insight into the conformational changes that accompany activation of a GPCR (see Gether 2000; Hubbell et al. 2000). Another breakthrough has been the improvements in our understanding of the GPCR tertiary structure. Low-resolution structures of rhodopsin based on cryo-electron microscopy of two-dimensional crystals resolved by Schertler et al. (Schertler et al. 1993; Schertler and Hargrave 1995) became available already a few years ago. These provided important insights into the organization of the transmembrane bundle and allowed the development of tertiary structure models of GPCRs (Ballesteros and Weinstein 1995; Scheer et al. 1996; Baldwin et al. 1997). Recently, Palczewski et al. (Palczewski et al. 2000) succeeded in generating three-dimensional crystals of rhodopsin for X-ray crystallography offering for the first time a tertiary structure model of a GPCR at atomic resolution (2.8 A) (Fig. 3.1). The structure appeared to be remarkably similar to the majority of existing receptor models that were developed based on the low-resolution structures, and accordingly a major revision of our current understanding of GPCR function has not been necessary. Nonetheless, the availability of high-resolution structural information has been able to clarify in particular previous hypothetical predictions regarding, for example, specific intramolecular interactions. Hence, it is now become possible to consider the functional roles of even individual side chains and pinpoint their specific role in receptor activation. This chapter will describe our current insight into agonist induced conforma-tional changes in the GPCR structure leading to activation of the G protein cascade and discuss the importance of constraining intramolecular interactions that keep the receptor predominantly inactive.

Fig. 3.1 The high-resolution structure of rhodopsin seen from the side (upperpanel) and from the top (lowerpanel) (Palczewski et al. 2000). The seven transmembrane helices (indicated by numbers) are organized in a counterclockwise fashion (as seen from the extracellular side in the lower panel). A buried ligand binding crevice containing the covalently attached chromophore, retinal, is formed between the seven helices. The second extracellular loop 2 (ECL2, show in green) dives into the transmembrane domain and forms a plug in the binding crevice. (See Plate 2.)

Fig. 3.1 The high-resolution structure of rhodopsin seen from the side (upperpanel) and from the top (lowerpanel) (Palczewski et al. 2000). The seven transmembrane helices (indicated by numbers) are organized in a counterclockwise fashion (as seen from the extracellular side in the lower panel). A buried ligand binding crevice containing the covalently attached chromophore, retinal, is formed between the seven helices. The second extracellular loop 2 (ECL2, show in green) dives into the transmembrane domain and forms a plug in the binding crevice. (See Plate 2.)

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