Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.

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Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor

Publisher Summary This chapter focuses on interleukin-8 (IL-8) and related chemotactic cytokines—namely, CXC and CC chemokines. IL-8 is the best known member of a new class of cytokines that are widely studied because of their ability to attract and activate leukocytes, and their potential role as mediators of inflammation. IL-8 was originally isolated from the culture supernatants of stimulated human blood monocytes and was identified as a protein of 72 amino acids with a molecular weight of 8383. The three-dimensional structure of IL-8 has been studied by nuclear magnetic resonance spectroscopy and X-ray crystallography. In concentrated solution, and on crystallization, IL-8 is present as a dimer. The first CC chemokine was identified after cloning by differential hybridization from human tonsillar lymphocytes and was termed LD78. The CC and CXC chemokines are similar in size and have an overall structure that is characterized by the two intrachain disulfide bonds, short N-terminal and long C-terminal sequences. It discusses the role of chemokines in pathology with skin inflammation because psoriasis was the first disease to be linked to overproduction of IL-8. Several independent studies document the occurrence of high levels of IL-8 in the synovial fluid of inflamed joints of patients with different forms of rheumatic diseases, osteoarthritis, and gout.

Interleukin-8 and related chemotactic cytokines--CXC and CC chemokines.

A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model.

The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.

Sequence alignment of the G-protein coupled receptor superfamily.

LIGHT absorption by the visual pigment rhodopsin1,2 triggers, through G-protein coupling, a cascade of events in the outer segment of the rod cell of the vertebrate retina that results in membrane hyperpolarization and nerve excitation3–5. Rhodopsin, which contains 348 amino acids6–8, has seven helices that cross the disk membrane6,9 and its amino terminus is extracellular. A wealth of biochemical data is available for rhodopsin: 11-cis retinal is bound10 to lysine 296 in helix VII; glutamic acid 113 on helix III is the counterion to the protonated Schiff's base11,12; a disulphide bridge, cystine 110–187, connects helix III to the second extracellular loop e2 (refs 13, 14); the carboxy terminus has two palmitoylated cysteines forming a cytoplasmic loop i4 (ref. 15); three intracellular loops i2, i3 and i4 mediate activation of the heterotrimeric G protein transducin16,17; glutamic acid 135 and arginine 136 at the cytoplasmic end of helix III affect binding of transducin18. But to provide a framework to interpret these data, not only for rhodopsin but for other G-protein-coupIed receptors, requires the structure to be determined. Here we present a projection map of rhodopsin showing the configuration of the helices.

Projection structure of rhodopsin.

Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Gt). These are (i) the second cytoplasmic loop, which connects rhodopsin helices III and IV, (ii) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iii) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MII" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin II, exhibiting Kd values in the 2 microM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 microM. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.

J H McDowell

Papers

Three cytoplasmic loops of rhodopsin interact with transducin