Based on mutagenesis studies and molecular dynamics simulations on the GnRH receptor and P2-AR, Ballesteros et al. (46,63) suggested that the conserved R3.50 of the DRY motif is constrained by an ionic interaction with the preceding D3.49 in inactive forms. The crystal structure of inactive rhodopsin suggests that the carboxylate group of E3.49(134) forms a salt-bridge with the guanidium group of R3.50(135) (ref. 3; Fig. 4A). Therefore, it is inferred that disruption of the interaction between D/E3.49 and R3.50 would lead to activation of the receptors, mimicking protonated states of D/E3.49.

However, charge-neutralizing mutations at D/E3.49 in some 7TMRs, including those in the m1 muscarinic (64) and luteinizing hormone/chorionic gonadotropin (65) receptors, did not lead to enhanced constitutive activity. The mutation in the GnRH receptor did (46) or did not (66) lead to constitutive activation. The mutations reduced cell-surface expression of the receptors, suggesting that D/E3.49 is important for proper folding and, hence, the stability of the receptor proteins. Mutations at 6.34(X3) Sites

It is noteworthy that the residues at the 6.34 site within the X16.30BBX2X36.34B motif among rhodopsin-like 7TMRs vary markedly (Fig. 3B). Therefore, a direct interaction between R3.50 and the 6.34 site may not always be present in the receptors as it is in rhodopsin. However, mutations at the 6.34(X3) site or its variants at the junction of the third intracellular (i3) loop and TM6 have been shown to lead to constitutive activation of several 7TMRs, including A293 in a1B-AR (36), T373 in a2A-AR (67), L322 in p1-AR (68), C322 in the 5-HT2A receptor (69), S312 in the 5-

Fig. 4. (From opposite page) Activation of the b2-AR involves disruption of an ionic lock between TM3 and TM6. The Ca traces are taken from the high resolution structure of bovine rhodopsin. Except in A, the top of each panel shows the extracellular end, and the bottom of each panel shows the intracellular end of the TMs. (A) An extracellular view of the high resolution structure of rhodopsin showing the interaction between residues at the cytoplasmic ends of TM3 and TM6. (B) A side view of the interactions between E6.30 and R3.50 as well as between R3.50 and D3.49, which are within the distance range of an ionic interaction shown with dashed lines, in a rhodopsin-based model of the b2-AR. (C) After the E6.30A mutation, the ionic interaction of E6.30 and R3.50 is abolished. (Modified from Fig. 5 of Ballesteros et al., ref. 63.)

HT2C receptor (70), and T313 in the 5-HT1B receptor (71) (see Fig. 3B). In the a1B-AR, all 19 possible amino acid substitutions at the 6.34(X3) locus (A293) led to varying levels of constitutive activities, with the A6.34(293)K mutant demonstrating the highest activity (36).

The effect of a mutation at the 6.34 locus can be dramatically different depending on the nature of the substitution (44). For the ^-opioid receptor (Fig. 5), T6.34(279)K mutant dramatically enhanced agonist-independent activity, whereas T6.34(279)D mutation did not, although it almost abolished the G protein signaling (44). The results were interpreted in the structural context of a rhodopsin-based model for the ^-opioid receptor. The interaction of T6.34(279) with R3.50(165) through a hydrogen bond in the ^-opioid receptor stabilizes the inactive conformations (Fig. 6A). The T6.34(279)K substitution disrupts this interaction because of charge repulsion and supports agonist-free activation (Fig. 6B), whereas T6.34(279)D mutation strengthens this interaction by forming an even stronger ionic bond that keeps the receptor in inactive states (ref. 44; Fig. 6C).

However, introducing an acidic residue in the 6.34(X3) locus does not always lead to inactive receptors. In contrast to the ^-opioid receptor, substitutions of the 6.34(X3) locus with D or E caused agonist-independent activation of several 7TMRs, including A293D/E in the a1B-AR (36), T373E in the a2A-AR (67), L322E in the p1-AR (68) and C322E in the 5-HT2A receptor (69). The differences between those receptors and the ^-opioid receptor have been further studied and have been demonstrated to involve a mutation at the 6.30(X1) locus as shown in Subheading Neutralizing Mutations at Asp/Glu6.30(X1) Sites

Ballesteros et al. (2001) (63) observed that charge-neutralizing mutations (alanine substitution) of E6.30(268)(X1) alone or combined with that of Asp3.49(130) led to agonist-independent activities of the P2-AR, suggesting that the ionic interactions of E6.30(X1) with R3.50 in the inactive state (Fig. 4B) were disrupted by the mutations at these sites (Fig. 4C) (63). Although D/E6.30 is not universally conserved among the rhodopsin-like receptors, it is nearly 100% conserved among the monoamine and glycoprotein hormone receptors and opsins (63). Consistent with the observations regarding the P2-AR, the E6.30(360)A mutant of the m1 muscarinic receptor displays high agonist-independent activity (72). Based on computational modeling, the E6.30-R3.50 salt-bridge was proposed to occur in the 5-HT2A receptor (5,73,74) and the a1B-AR (75); this was supported by studies showing that mutations weakening this interaction led to constitutive activation. Additionally, naturally occurring D6.30(564)G mutation in the lutropin/ choriogonadotropin receptor (76,77) and D6.30(619)G mutation in the thy-

Rat \i opioid receptor

Rat \i opioid receptor

Fig. 5. Helical net representation of the rat m-opioid receptor. The D/ERY motif and X1BBX2X3B motif are highlighted with dark circles at the cytoplasmic ends of TM3 and TM6, respectively.

rotropin receptor (78) leads to constitutive receptor activation, which causes gonadotropin-independent familial male-limited precocious puberty and hyperfunctioning thyroid adenomas, respectively. The activation mechanism of the D6.30(564) mutant of the lutropin/choriogonadotropin receptor has been suggested to result from the loss of R3.50(464)-D6.30(564) ionic interaction (79). The finding that substitution of D6.30(564) with Glu did not lead to constitutive activation (77,80) is consistent with the interpretation. Therefore, the D/E6.30(X1)-R3.50 interaction may constitute a constraint of inactive states for many 7TMRs, which is removed in the process of receptor activation (73-75,79).

0 0

Post a comment