GPCR activation involves disruption of stabilizing intramolecular interactions

The biophysical studies together with an array of different indirect approaches have provided evidence for movements of TM6 relative to TM3 in agonist-induced receptor activation. But what are the molecular mechanisms that control the movements of TM6 and thus govern the transition of the receptor between its inactive and active state? An important discovery in relation to this question was the now well-established fact that many GPCRs possess basal activity and accordingly can activate the G protein in the absence of agonists (Costa and Herz 1989; Samama et al. 1993; Chidiac et al. 1994). In addition, discrete mutations are able to dramatically increase this constitutive agonist-independent receptor activity (Allen etal. 1991; Kjelsberg etal. 1992; Lefkowitz etal. 1993; Samama etal. 1993; Scheer et al. 1996). A crucial clue about the molecular mechanisms underlying constitutive receptor activation came from a study where the naturally occurring Ala2936.34 residue in the C-terminal part of third intracellular loop of the a1b adrenergic receptor was substituted with all other possible residues. They found that the presence of all other residues than the alanine resulted in higher agonist-independent receptor activity (Kjelsberg et al. 1992). This led to the suggestion that constraining intramolecular interactions have been conserved during evolution to maintain the receptor preferentially in an inactive conformation in the absence of agonist. Conceivably, these inactivating constraints could be released as a part ofthe receptor activation mechanism, either following agonist binding or due to specific mutations, causing key sequences to be exposed to G protein. The hypothesis has been indirectly supported by the observation that constitutively activated p2 adrenergic receptor and histamine H2 receptor mutants are characterized by a marked structural instability (Gether et al. 1997a; Rasmussen et al. 1999; Alewijnse et al. 2000) and enhanced conformational flexibility (Gether et al. 1997a). The data imply that the mutational changes have disrupted important stabilizing intramolecular interactions in the tertiary structure, allowing the receptor more readily to undergo conversion between its inactive and active state.

If receptor activation involves disruption of stabilizing intramolecular interactions, an obvious next question is which interactions are actually broken and how is this initiated following agonist binding to the receptor molecule. Despite the availability of high-resolution structure of a GPCR, this question can still not be fully answered; however, substantial evidence suggests that at least one of the key events in the activation process among Family 1 GPCRs involves protonation of the aspartic acid or glutamic acid in the highly conserved D/E RY (Glu/Asp-Arg-Tyr) motif at the cytoplasmic side of TM3 (Fig. 3.2 and see Chapter 1). It has been suggested that this protonation event leads to release ofconstraining intramolecular interactions, thereby resulting in movements of TM6 and conversion of the receptor to the active state. The most direct evidence for protonation of Asp/Glu3.49 has been obtained by Sakmar and coworkers (Arnis etal. 1994) who compared wild-type rhodopsin and rhodopsin mutated in position Glu1343.49 by flash photolysis allowing simultaneous measurement of photoproduct formation and rates of pH changes. Their data strongly suggested that proton uptake ofGlu1343.49 accompany formation of the metarhodopsin II state (Arnis etal. 1994). In the p2 adrenergic receptor lowering of the pH has also been shown to facilitate transition of the receptor to the activated state (Ghanouni et al. 2000). The 'protonation hypothesis' has been further supported by the observation that charge-neutralizing mutations, which mimics the protonated state of the aspartic acid/glutamic acid, cause dramatic constitutive activation of both the adrenergic a1b receptor, the p2 adrenergic receptor and the histamin H2 receptor (Scheer etal. 1996,1997; Rasmussen etal. 1999, Alewijnse etal. 2000). Similarly, improved coupling has been observed by mutation of the aspartic acid in the GnRH receptor (Ballesteros etal. 1998). Mutation of the aspartic residue in the M1 muscarinic receptor resulted in phosphoinositide turnover responses of the mutant that were quantitatively similar to the wild-type despite markedly lowered levels of expression (Lu etal. 1997). In parallel, constitutive activation was observed in rhodopsin following mutation ofthe glutamic acid found in the corresponding position of this receptor (Cohen et al. 1993). Finally, it was found that charge-neutralizing mutations of the aspartic acid (Asp1303.49) in the p2 adrenergic receptor are linked to the overall conformation of the receptor (Rasmussen et al. 1999). Thus, mutation of Asp1303.49 to asparagine did not only activate the receptor but also caused a cysteine in TM6 (Cys2856.47), which is not accessible in the wild-type receptor, to become accessible to methanethiosulfonate ethylammonium (MTSEA), a charged, sulfhydryl-reactive reagent (Rasmussen et al. 1999). This observation is consistent with a counter clockwise rotation (as seen from the extracellular side) and/or tilting of TM6 in the mutant receptor in agreement with the biophysical studies described above.

The network of constraining intramolecular interactions involving Asp/Glu3.49 and maintaining the receptor in its inactive state has until recently not been clear. Ballesteros et al. (1998) proposed that the ionic counterpart of Asp3.49 in the inactive state could be the adjacent Arg3.50 (Ballesteros et al. 1998) (Figs 3.2 and 3.4). In contrast, Scheer et al. (1996) have suggested, based on simulations in the a1B adrenergic receptor, that Arg3.50 in the inactive state is constrained in a 'polar pocket' formed by residues in TM1, 2, and 7 and that it is not interacting with Asp3.49 (Scheer et al. 1996). The specific ionic counterpart of the arginine in the inactive state in this scheme was predicted to be the conserved aspartic acid in TM2 (Asp2.50; Asp-79 in p2 adrenergic receptor) (Scheer et al. 1996). However, given that the high-resolution structure of rhodopsin indicates that Glu3.49 interacts with Arg3.50 in this receptor the most likely prediction is a similar interaction also among family A receptors containing an aspartate in position 3.49 (Fig. 3.4). In addition to the interaction with Glu3.49, the rhodopsin structure also suggested an interaction between Arg3.50 and a conserved glutamate in TM6, Glu6.30. A similar interaction was therefore proposed for the p2 adrenergic receptor and it was suggested that the interactions formed between Asp3.49, Arg3.50, and Glu6.30 form an ionic switch that controls transition of the receptor between its inactive and active state (Ballesteros et al. 2001a). The hypothesis was supported by the observation that charge-neutralizing mutation of Glu6.30 alone or in combination with Asp3.49 resulted in constitutive activation and a marked increased efficacy for the partial agonist pindolol (Ballesteros et al. 2001a) (Fig. 3.4). The mutants showed evidence that disruption of the interaction between TM3 and TM6 not only produced constitutive receptor activation but also demonstrated a tight correlation between the extent of constitutive activation and the extent of conformational rearrangement in TM6 as determined by assessing the accessibility of MTSEA to Cys2856.47 (Ballesteros etal. 2001a). Interestingly, substitution of Glu6.30 with alanine resulted in a higher degree of constitutive activation than substitution with glutamine (Fig. 3.4). In full agreement with the hypothesis, this is presumably because glutamine can still hydrogen bond to Arg3.50 and maintain an interaction, albeit more weakly (Ballesteros et al. 2001a).

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