Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.     

The hydrophobic amino acid residues in the membrane-proximal C tail of the G protein-coupled vasopressin V2 receptor are necessary for transport-competent receptor folding

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L(339), L(340)) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F(328), V(332) and L(336)) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S(330), S(331), S(333), S(334), S(338)) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport-competent folding state in the early secretory pathway.     

Three-layer ONIOM studies of the dark state of rhodopsin: the protonation state of Glu181

A computational three-layer ONIOM(QM-high:QM-low:MM) hybrid scheme has been applied to analyze the protonation state of the Glu181 amino acid residue in rhodopsin, which is vital to determining the rhodopsin photoactivation mechanism. Due to conflicting evidence from previous studies, it has yet to be conclusively resolved. In this study, we fully optimize dark-state rhodopsin model structures differing only at the 181-residue site—protonated and unprotonated Glu181—and calculate several experimentally observable properties. Comparison of calculated structures, excitation energies, and NMR chemical shifts for the two models with values from the literature allows a reevaluation of previously reported conclusions. A key finding is that the S₁ → S₂ energy level splitting, previously used as evidence for a neutral Glu181, is found to be almost identical for the two protonation states. We highlight a need for caution when interpreting experimental data. Small differences in the properties of the two model structures are also identified, which may be useful targets for future high-resolution experimental approaches.     

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G₁₁-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP β2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or β2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [³H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.     

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic “ionic lock.” Glu134^3.49 of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg135^3.50 in the dark-state crystal structure of rhodopsin. We examined the roles of Glu134^3.49 and Arg135^3.50 on H3 and Glu247^6.30 and Glu249^6.32 on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu134^3.49 of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg135^3.50 similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu247^6.30 and Glu249^6.32 on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu247^6.30 and Glu249^6.32 on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu134^3.49 and Arg135^3.50 is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the β₂-adrenergic receptor.     

Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice

Background

Membrane proteins constitute a major group of proteins and are of great significance as pharmaceutical targets, but underrepresented in the Protein Data Bank. Particular reasons are their low expression yields and the constant need for cautious and diligent handling in a sufficiently stable hydrophobic environment substituting for the native membrane. When it comes to protein crystallization, such an environment is often established by detergents.