Rhodopsin Activation Exposes a Key Hydrophobic Binding Site for the Transducin α-Subunit C Terminus

Janz, Jay M.; Farrens, David

doi:10.1074/jbc.m402567200

Cited by 103 publications

(158 citation statements)

References 59 publications

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“…2C Right). This type of interaction is consistent with experimental results that have already revealed a hydrophobic contribution to R*-G␣CT interaction (23). In the I-interaction, only 31% (892 Å 2 ) of the solvent accessible surface of ␣5 is excluded from the solvent.…”

Section: Kinetic Analysis: Intermediary Interaction Between Gt⅐gdp Ansupporting

confidence: 79%

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Scheerer¹,

Heck²,

Goede

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the ␣-subunit of intracellular G proteins (G␣␤␥). Kinetic analysis of transducin (Gt␣␤␥) activation shows that an intermediary R*⅐Gt␣␤␥⅐GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*⅐G protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G t␣ C-terminal peptide (G␣CT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal ␣-helix of Gt␣ (␣5) and shows how ␣5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them -termed stable or S-interaction -matches the position of the G␣CT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of G␣CT and conserved E(D)RY and NPxxY(x)5,6F regions of the GPCR. The alternative fit -termed intermediary or I-interaction -is distinguished by a tilt (42°) and rotation (90°) of ␣5 relative to the S-interaction and shows different ␣5 contacts with the NPxxY(x)5,6F region and the second cytoplasmic loop of R*. From the 2 ␣5 interactions, we derive a ''helix switch'' mechanism for the transition of R*⅐Gt␣␤␥⅐GDP to the nucleotide-free R*⅐G protein complex that illustrates how ␣5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.G protein coupled receptors (GPCRs) use the free energy of agonist binding to transmit physical or chemical signals into the cell. Bound agonists stabilize the 7 transmembrane (7TM) helix bundle of the receptor in an active conformation (R*). R* in turn interacts with intracellular heterotrimeric G proteins (G␣␤␥, G) to catalyze the exchange of GDP for GTP in the G␣ subunit and thus activate downstream effectors. According to classical receptor theory, R* is also stabilized by G protein binding (1). We used a synthetic peptide derived from the C terminus of the ␣-subunit of transducin (G␣CT), the key binding site for the receptor (2, 3), to stabilize and crystallize the R* conformation of opsin (4, 5). Opsin is the ligand-free form of the photoreceptor rhodopsin, and transducin (G t ␣␤␥, G t ) is its cognate G protein. X-ray structure analysis of the R*⅐G␣CT complex revealed that the cytoplasmic side of the TM5/TM6 helix pair (corresponding to the cytoplasmic loop C3 of the 7TM bundle) forms a mitt-like structure in which G␣CT is held. The contacts with R* induce in G␣CT an ␣-helical conformation with a C-terminal reverse turn (C-cap) (4, 6, 7), which is recognized by the receptor on the basis of its geometry (4).In GPCR mediated signal transduction, the signal is eventually established in the GTP-bound form of the G protein, which is the form that activates downstream effectors. The key step in whi...

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Section: Kinetic Analysis: Intermediary Interaction Between Gt⅐gdp Ansupporting

confidence: 79%

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Scheerer¹,

Heck²,

Goede

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The crystal structure of bovine rhodopsin shows that the cytoplasmic ends of helix V and VI are prominently extended below the plasma membrane in two different crystal forms, and these extended regions are highly flexible according to crystallographic temperature factors at more than 100 Å 2 (22,37). In rhodopsin, it has also been suggested that this region of helix V and the membrane-proximal region of helix VI function in activation of transducin via a carboxyl-terminal region of G␣ t (27,38,39).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Kuniyeda

Okuno

Terawaki

et al. 2007

Journal of Biological Chemistry

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. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTP␥S binding but intact inositol phosphate accumulation triggered by leukotriene B 4 stimulation. These results suggest that the i3-1 region is required only for G i activation. Moreover, in the i3-1 mutant, the deficiency in G i activation was accompanied by a loss of the high affinity leukotriene B 4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of G i proteins.Leukotriene B 4 (LTB 4 ) 3 is an inflammatory lipid mediator biosynthesized from membrane phospholipids by sequential enzymatic reactions (1-4). LTB 4 recruits and activates leukocytes, such as granulocytes, macrophages, monocytes, and T-cells, and plays pivotal roles in host defense and the pathogenesis of inflammatory and immune diseases (5, 6). These actions are mediated by two types of G-protein-coupled receptors (GPCRs). One is the LTB 4 receptor type-1 (BLT1), a high affinity LTB 4 receptor highly expressed in leukocytes (7). The other, BLT2, is a low affinity LTB 4 receptor expressed more ubiquitously than BLT1 in human tissues (8 -11).Many GPCRs can activate more than one G-protein subfamily member. For example, the prostaglandin E 2 receptor EP3D couples to G s and G o (12), platelet-activating factor receptor activates G i and G q (13,14), and the sphingosine 1-phosphate receptors S1P 1 and S1P 2 activate G i and G 12/13 (15). Previous reports suggest that in mediating different cellular events, BLT1 also utilizes different G-proteins, namely pertussis toxin (PTX)-sensitive G i/o and PTX-resistant G q subfamilies of G-proteins (7, 16). Promiscuous G-protein coupling enables GPCRs to mediate diverse signals.The molecular determinants of many receptors participating in G-protein coupling and activation have been investigated. With some exceptions, most of these studies emphasize the role of membrane-proximal regions of the second and third intracellular loops and of the cytoplasmic tail of the receptor (17). However, no consensus sequences for G-protein coupling have been clearly identified.We and others have reported that truncation of its cytoplasmic tail does not impair the ability of BLT1 to activate G-proteins (18,19). To identify regions of BLT1 responsible for G-protein coupling, we performed an extensive mutagenesis study of its intracellular domains. Here, we show that BLT1 distinguishes G i from G 16 at the amino terminus of the third intracellular region (the i3-1 region). Furthermore, Scatchard analyses of the i3-1 mutant revealed that BLT1 requires G i coupling to maintain a high affinity state for LTB 4 . A three-dimen-* Thi...

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“…By the significant rearrangement of loop C3 due to helix movement of TM5 and TM6, some residues in C3 are exposed, i.e. Leu-226, Phe-228 and Thr-229 on TM5, Ser-240 in C3, and Thr-242, Thr-243 and Met-253 on TM6 [127].…”

Section: Figure 38 "Ionic Lock" Of Opsin and Photoactivated Rhodopsmentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

857

925

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Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x) 5,6 F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

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Rhodopsin Activation Exposes a Key Hydrophobic Binding Site for the Transducin α-Subunit C Terminus

Cited by 103 publications

References 59 publications

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Identification of the Intracellular Region of the Leukotriene B4 Receptor Type 1 That Is Specifically Involved in Gi Activation

Crystal structure of the ligand-free G-protein-coupled receptor opsin

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