The G protein‐coupled receptor rhodopsin in the native membrane

Scheerer

Hofmann

et al. 2008

Nature

842

925

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.

Section: Overall Structure Of Opsinsupporting

confidence: 79%

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

Scheerer

Hofmann

et al. 2008

Nature

842

925

“…In addition, a growing body of evidence suggests that most GPCRs exist as oligomers in cellular membranes, complexes that are found in the endoplasmic reticulum and Golgi apparatus and thus occur during receptor maturation (reviewed by Bulenger et al (44)). Previously reported imaging data of native retinal disk membranes also suggest that certain receptor types, such as rhodopsin, are capable of organizing into higher order oligomers (45,46). However, the question as to whether G protein activation is dependent on oligomerization has yet to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

Efficient Coupling of Transducin to Monomeric Rhodopsin in a Phospholipid Bilayer

Whorton

Jastrzębska

Journal of Biological Chemistry

et al. 2008

Self Cite

241

179

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin⅐rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin⅐rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

“…Because most of the receptor molecule is unfolded out of the membrane during unfolding of this region, all of the molecular interactions with the rest of the receptor would be absent and the global stabilizing effects of Zn 2ϩ should be absent as well. Assuming a dimeric model of rhodopsin, the COOH-terminal region would be in contact with an adjacent rhodopsin molecule (50). Thus, the observed increase in force of this segment may derive from molecular interactions involving regions in the partner rhodopsin molecule.…”

Section: Unfolding Single Rhodopsin Molecules From Native Ros Discmentioning

confidence: 99%

“…6A) (50,51). Oligomerization of rhodopsin and other GPCRs has only recently become an appreciated concept and likely plays a central role in the signaling process (52).…”

Section: Unfolding Single Rhodopsin Molecules From Native Ros Discmentioning

confidence: 99%

Stabilizing Effect of Zn2+ in Native Bovine Rhodopsin

Journal of Biological Chemistry

Sapra

Koliński

et al. 2007

Self Cite

Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn 2؉ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl 2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl 2 , CdCl 2 , or CoCl 2 . Thus, Zn 2؉ stabilizes the structure of rhodopsin in a specific manner.Zinc is the second most abundant trace element found in the human body (1) and occurs in high concentrations in the retina (2). Zinc deficiency in humans can lead to vision-related disorders such as poor dark adaptation, night blindness, and retinal degeneration (3). The role of Zn 2ϩ in vision is likely related, in part, to rhodopsin, the light receptor that initiates phototransduction in the rod outer segments (ROS) 5 of retinal photoreceptor cells (4). Rhodopsin can directly associate with this bivalent metal ion (5, 6), and Zn 2ϩ increases the level of rhodopsin phosphorylation (7). Furthermore, changes are observed in the pattern of thermal bleaching and regeneration of rhodopsin by 11-cis-retinal in the presence and absence of Zn 2ϩ (6,8). Therefore, Zn 2ϩ appears to affect the structure and function of rhodopsin.Rhodopsin in native ROS disc membranes has been previously characterized by single-molecule force spectroscopy (SMFS) to reveal the molecular interactions that stabilize the dark state of the receptor (9). The receptor is organized into several stable structural segments that present barriers to unfolding. Forces required to unfold these segments provide a direct measure of the molecular interactions stabilizing the protein in a particular region. SMFS has also been used to characterize the molecular interactions underlying the stability of a few other membrane proteins including bacteriorhodopsin and halorhodopsin from Halobacterium salinarium (10, 11), the sodium/proton antiporter NhaA from Escherichia coli (12), and human aquaporin-1 (13). The sensitivity of SMFS assays enables one to probe the effects of environmental factors like temperature, pH, ion concentration, and oligomeric assembly on the molecular interactions stabilizing a protein (12, 14 -16).In the current study, we use SMFS to monitor the effect of Zn 2ϩ on molecular interactions stabilizing dark-state rhodopsin. Force-distance (F-D) curves obtained from native bovine ROS disc membranes in the presence of Zn 2ϩ revealed that the location of stable structural segments in rhodopsin is unaffected by the inclusion of this bivalent metal ion. However, forces required to unfold these segments in the presence of Zn 2ϩ were significantly increased. Thus, Zn 2ϩ appears to strengthen the molecular interactions that stabilize the native structure of rhodopsin. EXPERIMENTAL PROCEDURESROS Disc...