Introduction

1.1. Seven-Transmembrane Receptors

Seven-transmembrane receptors (7TMRs) comprise a large family of membrane-bound proteins that share a unifying signal transduction mechanism (i.e., upon activation, these receptors signal through G proteins). These receptors are involved in a vast variety of physiological functions, including neurotransmission, function of exocrine and endocrine glands, smell, taste, vision, chemotaxis, embryogenesis, development, human immunodeficiency virus (HIV) infection, oncogenesis, cell growth, and differentiation. More recent studies indicate that these receptors are also associated with and signal through other molecules (1). Therefore, it is more appropriate to use the term 7TMR than G protein-coupled receptors (GPCRs).

To date, rhodopsin is the only receptor of the superfamily for which the high-resolution structure has been determined (2,3). In 1993, Shertler et al. (2) published a project map of the bovine rhodopsin at 9 A resolution in two dimensions. In 2000, Palczewski et al. reported the structure of bovine rhodopsin in ground (inactive) states at 2.8 A resolution in three dimensions (3,4). Both reports showed the seven helices of rhodopsin traversing the plane of the membranes in a nonparallel manner, with some transmembranes (TMs) being tilted, an extracellular N-terminal domain, an intracellular C-terminal domain, and three extracellular loops and three intracellular loops connecting the helices (Fig. 1). In the proximal region of the C-terminal

From: Contemporary Clinical Neuroscience: The G Protein-Coupled Receptors Handbook Edited by: L. A. Devi © Humana Press Inc., Totowa, NJ

Fig. 1. Three-dimensional crystal structure of rhodopsin with bound detergent and amphiphile molecules. Helical portions of the protein, including the seven TMs, are shown as blue rods, and P-strands are shown as blue arrows. The polypeptide connecting the helices appears as blue coils. A transparent envelope around the protein represents the molecular surface. The dark blue ball-and-stick groups at the bottom of the figure denote carbohydrate groups attached to the protein. Two palmitoyl groups covalently attached to the protein are shown in green. Nonylglucoside and heptanetriol molecules located near the hydrophobic surface of the protein are shown in yellow. (Reprinted from ref. 4 with permission of the American Chemical Society, copyright 2001.)

Fig. 1. Three-dimensional crystal structure of rhodopsin with bound detergent and amphiphile molecules. Helical portions of the protein, including the seven TMs, are shown as blue rods, and P-strands are shown as blue arrows. The polypeptide connecting the helices appears as blue coils. A transparent envelope around the protein represents the molecular surface. The dark blue ball-and-stick groups at the bottom of the figure denote carbohydrate groups attached to the protein. Two palmitoyl groups covalently attached to the protein are shown in green. Nonylglucoside and heptanetriol molecules located near the hydrophobic surface of the protein are shown in yellow. (Reprinted from ref. 4 with permission of the American Chemical Society, copyright 2001.)

domain, there is a short helix, helix 8 (H8), parallel to the plane of the plasma membranes (Fig. 1). It is generally accepted that 7TMRs share the structure of the 7-TM bundle (5,6).

In the human genome, there are three major families of 7TMRs (see refs. 7 and 8 for a classification scheme): (a) rhodopsin and rhodopsin-like receptors (approx 200) and odorant and taste receptors (several hundreds); (b) glucagons/vasoactive intestinal polypeptide/calcitonin receptors (approx 25); and (c) metabotropic glutamate receptors, y-aminobutyric acid (GABA)b receptors and chemosensors (approx 20). Within each family, there is at least 25% homology within the 7-TMs and a distinctive set of highly conserved residues and motifs. The rhodopsin family of 7TMRs, which constitute approx 90% of all 7TMRs (4), are the most extensively studied.

1.2. Numbering Schemes for Rhodopsin-Like 7TMR Sequences

Two residue-numbering schemes are used throughout this chapter: the generic numbering scheme of Ballesteros and Weinstein (9) and the residue numbers in the amino acid sequence of the particular receptor being discussed. According to the generic nomenclature, amino acid residues in TMs are assigned two numbers (N1 and N2). N1 refers to the TM number. For N2, the most conserved residue in each TM is assigned 50, and the other residues are numbered in relation to this conserved residue, with numbers decreasing toward the N-terminus and increasing toward the C-terminus. The receptors of the rhodopsin family are characterized by the presence of highly conserved "fingerprint" residues (8,10), including N1.50 in the TM1; D2.50 in the TM2; the DRY(3.49-3.51) motif in the TM3; W4.50 in the TM4; and P5.50, P6.50, and NP7.50XXY motif in TMs5, -6, and -7.

For example, D3.49(134) in bovine rhodopsin is located in TM3 and one amino acid N-terminal to the most conserved R3.50; it is the 134th amino acid residue from the N-terminus. The generic numbering scheme allows easy comparisons of the same residues among different receptors in the rhodopsin family.

1.3. Structural Studies on Rhodopsin

The structure-function relationship of rhodopsin has been extensively studied (reviewed in refs. 11-13) with low-resolution electron density maps (2,14,15) and biochemical approaches, including crosslinking (by genetically engineered disulfide bridges and Zn2+ binding sites), site-directed spin labeling, scanning accessibility determinations (reviewed in ref. 11), and analysis of retinal movement by photo-affinity labeling (16). By X-ray crystallography, Palczewski et al. reported a high-resolution structure of the bovine rhodopsin in ground (inactive) states at 2.8 A resolution (3,4), which

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