The VIYIMMNK sequence (boxed words) represents the epitope of the TM7C antibody and incorporates a part of one of the most conserved motifs (NPXXY) in the family of G protein-coupled receptors

The VIYIMMNK sequence (boxed words) represents the epitope of the TM7C antibody and incorporates a part of one of the most conserved motifs (NPXXY) in the family of G protein-coupled receptors. Recently, there has been considerable progress in understanding retinalprotein interactions and the structural rearrangements that the protein undergoes uponcistransisomerization of the Bisacodyl chromophore (12,13). significantly at the metarhodopsin III intermediate. Further, incubation of the antigenantibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling. Rhodopsin, a member of a large family of seven-transmembrane (TM) helix receptors (Fig.1), triggers a light-dependent, phosphodiesterase-mediated, cyclic guanosine monophosphate-regulated, and G protein-coupled signal transduction cascade (17). Rhodopsin contains 11-cis-retinal covalently bound via a protonated Schiff base (PSB) to the -amino group of Lys-296 on TM7. The carboxyl group of Glu-113 on TM3, which resides well within the membrane, neutralizes the positive charge on the retinyl-PSB in the dark state (810). This dominant electrostatic interaction and the steric constraint imposed by the very nature of the chromophore in its immediate protein environment supports the inactive ground-state conformation. Upon light absorption, retinal isomerizes to the all-transconfiguration that drives the protein through a series of transient and thermally stable intermediates (11). Charge separation between the retinyl-PSB and Glu-113 with subsequent protonation of the latter culminates in formation of metarhodopsin II (Meta II), an active intermediate that accommodates the high-affinity sites for interaction with several regulatory proteins of the visual transduction cascade. == Figure 1. == Secondary structure model of bovine rhodopsin. The transmembrane helices are numbered IVII from the amino to the carboxyl terminus. Asn-2 and Asn-15 areN-glycosylated, Cys-110 and Cys-187 are linked by a disulfide bond, and Cys-322 and Cys-323 are palmitoylated. Exposed cysteine residues on the second (Cys-140) and fourth (Cys-316) cytoplasmic loops are indicated. Lys-296, the site of attachment of retinal, and Glu-113, the counterion to Bisacodyl the protonated retinyl-Schiff Bisacodyl base, also are indicated. The VIYIMMNK sequence (boxed letters) represents the epitope of the TM7C antibody and incorporates a part of one of the most conserved motifs Rabbit Polyclonal to DNA-PK (NPXXY) in the family of G protein-coupled receptors. Recently, there has been considerable progress in understanding retinalprotein interactions and the structural rearrangements that the protein undergoes uponcistransisomerization of the chromophore (12,13). Despite these advances, a clear understanding of how changes deep in the membrane are relayed to the surface of the protein, thereby allowing an opening of its conformation for interaction with the signaling machinery, remains largely unknown. It is evident that a Bisacodyl comprehensive understanding of how a receptor communicates with this machinery will require the identification of light- or agonist-induced conformational changes in molecular detail. To this end, single and double cysteine-substitution mutants of rhodopsin in conjunction with site-directed spin labeling and electron paramagnetic resonance spectroscopy have provided valuable information about both the dark- and light-activated states (1421). Further, metal-binding sites or disulfide bonds have been engineered between the TM helices to restrain possible light-induced conformational changes at specific locations in rhodopsin (22,23). Two important conclusions to arise from these studies are that the cytoplasmic termini of TM3 and TM6 are close in proximity and that the light-induced movement of these helices relative to each other is required to adopt an active conformation. In the present paper, we show by using an antirhodopsin mAb that light induces the exposure of an epitope that extends from the region between Lys-296 and the cytoplasmic end of TM7. Furthermore, we demonstrate that this region of the protein, which contains the highly conserved NPXXY motif implicated in signaling and agonist-induced internalization of several G protein-coupled receptors (2426), becomes accessible to the antibody exclusively at the Meta II stage of activation. == MATERIALS AND METHODS == == Materials. == Protein A and Con A Sepharose were purchased from Pharmacia. The hybridoma isotyping kit and the alkaline phosphatase-conjugated goat anti-mouse IgG were from Calbiochem. Horseradish peroxidase-conjugated goat anti-mouse IgG was from Promega. ELISA plates were from Nunc, and polyvinylidene fluoride transfer membranes were from Millipore. Peptides corresponding to the carboxyl-terminal region of TM7 were synthesized at the peptide synthesis facilities of the Max Planck Institute for Biophysics (Frankfurt). Bovine retinae were from W. L. Lawson (Lincoln, NE), and 11-cis-retinal was a gift of R. Crouch (Medical University of South Carolina and the National Eye Institute). The A5 mAb, which is specific for the amino-terminal sequence of bacteriorhodopsin, has been described (27). Other materials used in this investigation have been described (28). == Preparation of Rod Outer Segments and Disk Membranes. == Bovine rod outer segments.