Photoreceptor preservation induced by intravitreal controlled delivery of GDNF and GDNF/melatonin in rhodopsin knockout mice.

Purpose: To evaluate the potential of a poly(lactic-co-glycolic acid) (PLGA)-based slow release formulation of glial cell line-derived neurotrophic factor (GDNF) alone or in combination with melatonin to rescue photoreceptors in a mouse model of retinal degeneration. Methods: GDNF and GDNF/melatonin-loaded PLGA microspheres (MSs) were prepared using a solid-in-oil-in-water emulsion solvent extraction-evaporation technique. A combination of PLGA and vitamin E (VitE) was used to create the microcarriers. The structure, particle size, encapsulation efficiency, and in vitro release profile of the microparticulate formulations were characterized. Microparticulate systems (non-loaded, GDNF, and GDNF/melatonin-loaded MSs) were administered intravitreally to 3-week-old rhodopsin knockout mice (rho (-/-); n=7). The functional neuroprotective effect was assessed with electroretinography at 6, 9, and 12 weeks old. The rescue of the structure was determined with photoreceptor quantification at 12 weeks (9 weeks after administration of MSs). Immunohistochemistry for photoreceptor, glial, and proliferative markers was also performed. Results: The microspheres were able to deliver GDNF or to codeliver GDNF and melatonin in a sustained manner. Intravitreal injection of GDNF or GDNF/melatonin-loaded MSs led to partial functional and structural rescue of photoreceptors compared to blank microspheres or vehicle. No significant intraocular inflammatory reaction was observed after intravitreal injection of the microspheres. Conclusions: A single intravitreal injection of GDNF or GDNF/melatonin-loaded microspheres in the PLGA/VitE combination promoted the rescue of the photoreceptors in rho (-/-) mice. These intraocular drug delivery systems enable the efficient codelivery of therapeutically active substances for the treatment of retinal diseases.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

The Energetics of Chromophore Binding in the Visual Photoreceptor Rhodopsin.

The visual photoreceptor rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that stabilizes its inverse agonist ligand, 11-cis-retinal (11CR), by a covalent, protonated Schiff base linkage. In the visual dark adaptation, the fundamental molecular event after photobleaching of rhodopsin is the recombination reaction between its apoprotein opsin and 11CR. Here we present a detailed analysis of the kinetics and thermodynamics of this reaction, also known as the "regeneration reaction". We compared the regeneration of purified rhodopsin reconstituted into phospholipid/detergent bicelles with rhodopsin reconstituted into detergent micelles. We found that the lipid bilayer of bicelles stabilized the chromophore-free opsin over the long timescale required for the regeneration experiments, and also facilitated the ligand reuptake binding reaction. We utilized genetic code expansion and site-specific bioorthogonal labeling of rhodopsin with Alexa488 to enable, to our knowledge, a novel fluorescence resonance energy transfer-based measurement of the binding kinetics between opsin and 11CR. Based on these results, we report a complete energy diagram for the regeneration reaction of rhodopsin. We show that the dissociation reaction of rhodopsin to 11CR and opsin has a 25-pM equilibrium dissociation constant, which corresponds to only 0.3 kcal/mol stabilization compared to the noncovalent, tightly bound antagonist-GPCR complex of iodopindolol and ß-adrenergic receptor. However, 11CR dissociates four orders-of-magnitude slower than iodopindolol, which corresponds to a 6-kcal/mol higher dissociation free energy barrier. We further used isothermal titration calorimetry to show that ligand binding in rhodopsin is enthalpy driven with -22 kcal/mol, which is 12 kcal/mol more stable than the antagonist-GPCR complex. Our data provide insights into the ligand-receptor binding reaction for rhodopsin in particular, and for GPCRs more broadly.

Specific visible radiation facilitates lipolysis in mature 3T3-L1 adipocytes via rhodopsin-dependent ß3-adrenergic signaling.

The regulation of fat metabolism is important for maintaining functional and structural tissue homeostasis in biological systems. Reducing excessive lipids has been an important concern due to the concomitant health risks caused by metabolic disorders such as obesity, adiposity and dyslipidemia. A recent study revealed that unlike conventional care regimens (e.g., diet or medicine), low-energy visible radiation (VR) regulates lipid levels via autophagy-dependent hormone-sensitive lipase (HSL) phosphorylation in differentiated human adipose-derived stem cells. To clarify the underlying cellular and molecular mechanisms, we first verified the photoreceptor and photoreceptor-dependent signal cascade in nonvisual 3T3-L1 adipocytes. For a better understanding of the concomitant phenomena that result from VR exposure, mature 3T3-L1 adipocytes were exposed to four different wavelengths of VR (410, 505, 590 and 660nm) in this study. The results confirmed that specific VR wavelengths, especially 505nm than 590nm, increase intracellular cyclic adenosine monophosphate (cAMP) levels and decrease lipid droplets. Interestingly, the mRNA and protein levels of the Opn2 (rhodopsin) photoreceptor increased after VR exposure in mature 3T3-L1 adipocytes. Subsequent treatment of mature 3T3-L1 adipocytes at a specific VR wavelength induced rhodopsin- and ß3-adrenergic receptor (AR)-dependent lipolytic responses that consequently led to increases in intracellular cAMP and phosphorylated HSL protein levels. Our study indicates that photoreceptors are expressed and exert individual functions in nonvisual cells, such as adipocytes. We suggest that the VR-induced photoreceptor system could be a potential therapeutic target for the regulation of lipid homeostasis in a non-invasive manner.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state.

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

Structure-based simulations reveal concerted dynamics of GPCR activation.

G protein-coupled receptors (GPCRs) are a vital class of proteins that transduce biological signals across the cell membrane. However, their allosteric activation mechanism is not fully understood; crystal structures of active and inactive receptors have been reported, but the functional pathway between these two states remains elusive. Here, we use structure-based (Go-like) models to simulate activation of two GPCRs, rhodopsin and the ß2 adrenergic receptor (ß2AR). We used data-derived reaction coordinates that capture the activation mechanism for both proteins, showing that activation proceeds through quantitatively different paths in the two systems. Both reaction coordinates are determined from the dominant concerted motions in the simulations so the technique is broadly applicable. There were two surprising results. First, the main structural changes in the simulations were distributed throughout the transmembrane bundle, and not localized to the obvious areas of interest, such as the intracellular portion of Helix 6. Second, the activation (and deactivation) paths were distinctly nonmonotonic, populating states that were not simply interpolations between the inactive and active structures. These transitions also suggest a functional explanation for ß2AR's basal activity: it can proceed through a more broadly defined path during the observed transitions.

Precision vs flexibility in GPCR signaling.

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E(D)RY and Yx7K(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general.

Allosteric modulators of rhodopsin-like G protein-coupled receptors: opportunities in drug development.

Rhodopsin-like (class A) G protein-coupled receptors (GPCRs) are one of the most important classes of drug targets. The discovery that these GPCRs can be allosterically modulated by small drug molecules has opened up new opportunities in drug development. It will allow the drugability of "difficult targets", such as GPCRs activated by large (glyco)proteins, or by very polar or highly lipophilic physiological agonists. Receptor subtype selectivity should be more easily achievable with allosteric than with orthosteric ligands. Allosteric modulation will allow a broad spectrum of pharmacological effects largely expanding that of orthosteric ligands. Furthermore, allosteric modulators may show an improved safety profile as compared to orthosteric ligands. Only recently, the explicit search for allosteric modulators has been started for only a few rhodopsin-like GPCRs. The first negative allosteric modulators (allosteric antagonists) of chemokine receptors, maraviroc (CCR5 receptor), used in HIV therapy, and plerixafor (CXCR4 receptor) for stem cell mobilization, have been approved as drugs. The development of allosteric modulators for rhodopsin-like GPCRs as novel drugs is still at an early stage; it appears highly promising.

Molecular modeling of neurokinin B and tachykinin NK3 receptor complex.

The tachykinin receptor NK3 is a member of the rhodopsin family of G-protein coupled receptors. The NK3 receptor has been regarded as an important drug target due to diverse physiological functions and its possible role in the pathophysiology of psychiatric disorders, including schizophrenia. The NK3 receptor is primarily activated by the tachykinin peptide hormone neurokinin B (NKB) which is the most potent natural agonist for the NK3 receptor. NKB has been reported to play a vital role in the normal human reproduction pathway and in potentially life threatening diseases such as pre-eclampsia and as a neuroprotective agent in the case of neurodegenerative diseases. Agonist binding to the receptor is a critical event in initiating signaling, and therefore a characterization of the structural features of the agonists can reveal the molecular basis of receptor activation and help in rational design of novel therapeutics. In this study a molecular model for the interaction of the primary ligand NKB with its G-protein coupled receptor NK3 has been developed. A three-dimensional model for the NK3 receptor has been generated by homology modeling using rhodopsin as a template. A knowledge based docking of the NMR derived bioactive conformation of NKB to the receptor has been performed utilizing limited ligand binding data obtained from photoaffinity labeling and site-directed mutagenesis studies. A molecular model for the NKB-NK3 receptor complex obtained sheds light on the topographical features of the binding pocket of the receptor and provides insight into the biochemical data currently available for the receptor.

Structural insights into agonist-induced activation of G-protein-coupled receptors.

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the ß(2) adrenergic receptor (ß(2)AR) and the A(2A) adenosine receptor (A(2A)AR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8Å depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the ß(2)AR) or not (A(2A)AR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3-TM5-TM6 interface, and possibly constitutes a common theme of GPCR activation.

Conserved Tyr223(5.58) plays different roles in the activation and G-protein interaction of rhodopsin.

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I ⇌ Meta IIa ⇌ Meta IIb ⇌ Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.

The structural basis of agonist-induced activation in constitutively active rhodopsin.

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 Å for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal ß-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.

A conserved aromatic lock for the tryptophan rotameric switch in TM-VI of seven-transmembrane receptors.

The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seven-transmembrane (7TM) receptors. Molecular dynamics simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the beta(2)-adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated the constitutive signaling and strongly impaired agonist-induced signaling without affecting agonist affinity and potency. Ala substitution of Phe-V:13, the presumed interaction partner for Trp-VI:13, also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13 could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V:13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor activation.

Multiple switches in G protein-coupled receptor activation.

The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

Coarse-grained modeling of allosteric regulation in protein receptors.

Allosteric regulation provides highly specific ligand recognition and signaling by transmembrane protein receptors. Unlike functions of protein molecular machines that rely on large-scale conformational transitions, signal transduction in receptors appears to be mediated by more subtle structural motions that are difficult to identify. We describe a theoretical model for allosteric regulation in receptors that addresses a fundamental riddle of signaling: What are the structural origins of the receptor agonism (specific signaling response to ligand binding)? The model suggests that different signaling pathways in bovine rhodopsin or human beta(2)-adrenergic receptor can be mediated by specific structural motions in the receptors. We discuss implications for understanding the receptor agonism, particularly the recently observed "biased agonism" (selected activation of specific signaling pathways), and for developing rational structure-based drug-design strategies.

Evaluation of homology modeling of G-protein-coupled receptors in light of the A(2A) adenosine receptor crystallographic structure.

Homology modeling of the human A(2A) adenosine receptor (AR) based on bovine rhodopsin predicted a protein structure that was very similar to the recently determined crystallographic structure. The discrepancy between the experimentally observed orientation of the antagonist and those obtained by previous antagonist docking is related to the loop structure of rhodopsin being carried over to the model of the A(2A) AR and was rectified when the beta(2)-adrenergic receptor was used as a template for homology modeling. Docking of the triazolotriazine antagonist ligand ZM241385 1 was greatly improved by including water molecules of the X-ray structure or by using a constraint from mutagenesis. Automatic agonists docking to both a new homology modeled receptor and the A(2A) AR crystallographic structure produced similar results. Heterocyclic nitrogen atoms closely corresponded when the docked adenine moiety of agonists and 1 were overlaid. The cumulative mutagenesis data, which support the proposed mode of agonist docking, can be reexamined in light of the crystallographic structure. Thus, homology modeling of GPCRs remains a useful technique in probing the structure of the protein and predicting modes of ligand docking.

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors.

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in beta(2)-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135(3.50) at the intracellular end of TM3 (part of the DRY motif) and E247(6.30) on TM6, and the rotamer toggle switch on W265(6.48) on TM6. We observe that the toggling of the W265(6.48) rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298(7.45), W265(6.48) and H211(5.46). As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139(3.54), T229, Q237, Q239, S240, T243 and V250(6.33), become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139(3.54), T229 and V250(6.33), are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265(6.48) and H211(5.46) and E122(3.37) and C167(4.56). These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors.

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist.

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.

G protein coupled receptor structure and activation.

G protein coupled receptors (GPCRs) are remarkably versatile signaling molecules. The members of this large family of membrane proteins are activated by a spectrum of structurally diverse ligands, and have been shown to modulate the activity of different signaling pathways in a ligand specific manner. In this manuscript I will review what is known about the structure and mechanism of activation of GPCRs focusing primarily on two model systems, rhodopsin and the beta(2) adrenoceptor.