TM7 and the NPxxY Motif

The high conservation of the NP*xx(x)Y motif in the rhodopsin family receptors is reflected in the reference position for rhodopsin of Pro303750, which is identified in the motif by an asterisk. Like all of the TM helices, except TM3, helix TM7 is not contiguous and is kinked. The location of the kink is maintained by Cys264647, which acts like a peg upon which the TM7 helix is hung and closes a circle of interaction with retinal (Figure 10.6A). The g-sulfur of Cys264647 lies adjacent to Trp265648, part of the highly conserved TM6 CWxP* motif, which provides an interactive surface for the cyclohexenyl ring of retinal. The polyene chain of the chromophore is attached to Lys296743, one helical turn upstream from the kink in TM7 which is pegged down by Cys264647. The stretch of helix between Lys296743 and the kink adopts a 310 configuration.

The importance of Asn302749 is revealed by its interaction with Asp250 in the core of the helical bundle. Intriguingly, the strict conservation of these residues is

FIGURE 10.6 The conserved dithiol bridge and b4-strand fasten retinal to TM3. A, rhodopsin molecule B, sliced with a slabbing plane parallel to helix 3 (cf. figure 10.3A), reveals the polyene tail aligned with and extending the b-sheet. Lateral location of the ring directly by 3 Cys167 (g-sulfur is shown) and through 6 Trp265 indirectly by 6 Cys264, acting as a pivot underneath which helix 7 (indicated by the thin line) is bent. B, the sulfur-aromatic cage fastening retinal to helix 3, with Tyr (darker atomic bonds) near the b-strands and mainly Phe (lighter atomic bonds) in the center of the bilayer, extends to incorporate 7 Lys296 (ball-and-stick). Helix 3 is contiguous from Cys110 to Arg135 (ball-and-stick).

FIGURE 10.6 The conserved dithiol bridge and b4-strand fasten retinal to TM3. A, rhodopsin molecule B, sliced with a slabbing plane parallel to helix 3 (cf. figure 10.3A), reveals the polyene tail aligned with and extending the b-sheet. Lateral location of the ring directly by 3 Cys167 (g-sulfur is shown) and through 6 Trp265 indirectly by 6 Cys264, acting as a pivot underneath which helix 7 (indicated by the thin line) is bent. B, the sulfur-aromatic cage fastening retinal to helix 3, with Tyr (darker atomic bonds) near the b-strands and mainly Phe (lighter atomic bonds) in the center of the bilayer, extends to incorporate 7 Lys296 (ball-and-stick). Helix 3 is contiguous from Cys110 to Arg135 (ball-and-stick).

violated by the GnRH receptor, in which the consensus aspartate Asp2 50 is an asparagine and Asn749 is an aspartate. The GnRH receptor is a minimalist 7TMR and is unusually short; helix TM1 is a retained signal peptide and sequence termination immediately at the end of the TM7 removes cysteine palmitoylation and desensitizing phosphorylation sites. Investigation had confirmed that this was a mutually compensating double substitution before the crystal structure of rhodopsin was known.35 "Restoration" of the conserved Asp250 was detrimental to binding and activation with GnRH, which was recoverable when the double substitution was made by "restoration" of Asn749 as well.

The highly conserved Tyr296753 is usually overlooked in most sequence-structure comparisons, although preoccupation with the non-conservation of the NPxxY motif in the GnRH receptor also led to SDM studies at this position. The importance of the phenyl ring was revealed by a Y332A receptor mutant that bound ligand but demonstrated no functional activity, whereas a Y332F mutant behaved like the wildtype receptor.36

It is not usually realized that the NPxx(x)Y motif is also a phosphorylation acceptor site.37 Because it lies between helix 7 as it exits the membrane and helix 8 lying parallel to the membrane and is accessible to an antibody after photoacti-

vation of rhodopsin,38 then kinase interaction and phosphorylation of this interhelical loop is a feasible proposition. Surprisingly few studies have addressed the role of the tyrosine experimentally, although most recently an alanine substitution was shown to produce a biologically inactive B2 bradykinin receptor, which was con-stitutively phosphorylated and internalized.39

The substantial movement of the intracellular ends of the TM helices, moving 28 A nearer together, was already evident in the colocalization of helices TM3 and TM7 after photoactivation, allowing a cystine bridge to form (Figure 10.4). The Tyr296753 position between helices 7 and 8 is separated from Arg135350 at the carboxyl terminus of TM3 by only one hydrophobic reside. The arginine of the TM3 DRY motif was one of the first residues implicated in signal transmission for both the rhodopsin and the secretin receptor families.

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