Conformational changes involved in G-protein-coupled-receptor activation

Abstract

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M(3) muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.

Figure 1

Agonist-modulated disulfide cross-linking in double Cys mutant M₃ mAChRs. All Cys substitutions were introduced into the M3’(3C)-Xa receptor background, a modified version of the rat M₃ mAChR [18]. In this construct, most endogenous Cys residues were replaced with either serine or alanine (boxed residues). The M3'(3C)-Xa construct contains only three remaining native Cys residues, C140, C220, and C532^7.42 (note that C140 and 220 are engaged in a disulfide bond). In addition, the central portion of the i3 loop (A274-K469) was replaced with two adjacent factor Xa cleavage sites. The five potential N-glycosylation sites present in the N-terminal portion of the receptor protein (N6, N15, N41, N48, and N52) were replaced with Gln residues (not shown). A rabbit polyclonal antibody (referred to as anti-C-M3) was raised against the indicated C-terminal receptor sequence [18]. Muscarinic agonists promoted the formation of disulfide cross-links between Cys residues present at positions 151^3.36 and 532^7.42 (red rings; [21]), 88^1.53 and 543^7.53 (brown rings; [20]), 91^1.56 and 545^7.55/546^7.56 (orange rings; [20]), 169^3.54 and 484^6.29/488^6.33 (purple rings; [22]), 250C^5.58 and 491C^6.36/492^6.37 (light blue rings; [24]), 253^5.61 and 489^6.34-492^6.37 (green rings; [24]), and 254^5.62 and 489^6.34-492^6.37 (green rings; [18]). In contrast, muscarinic agonists inhibited the formation of disulfide cross-links between Cys residues present at positions 91^1.56/92^1.57 and 549^7.59/550^7.60 (dark blue rings) [23]. The residues highlighted by red letters are known to play key roles in the binding of ACh and other conventional muscarinic agonists [33, 34]. Numbers refer to amino acid positions in the rat M₃ mAChR sequence [33]. I-VII, TM helices I-VII; i1-i3, the three intracellular loops of the M₃ mAChR; H8, helix 8.

Figure 2

General strategy used to detect the formation of disulfide bonds between vicinal Cys residues in mutant M₃ mAChRs. Pairs of Cys residues were introduced into the M3’(3C)-Xa background receptor (see Figure 1), one Cys N-terminal and the other one C-terminal of the factor Xa cleavage site. (a, c) When two Cys residues lie adjacent to each other in the 3D structure of the receptor, they have the potential to form a disulfide bridge, either spontaneously or in the presence of oxidizing agents. Following cleavage with factor Xa, the two resulting receptor fragments will remain covalently linked by the disulfide bridge. Consequently, a full-length receptor band (~38 kDa in size) can be detected on Western blots run under non-reducing conditions (e, left lane). (b, d) When the two introduced Cys residues are not close to each other in the 3D structure of the receptor, they are unable to form a disulfide bridge. In this case, factor Xa digestion will yield two separate cleavage products and a full-length receptor band will not appear on Western blots run under non-reducing conditions (e, right lane).

Figure 3

Views of the M₃ mAChR depicting regions/amino acids that undergo activity-dependent conformational changes. A 3D model of the inactive state of the rat M₃ mAChR was built via homology modeling using the high-resolution X-ray structure of bovine rhodopsin as a template [2, 20]. (a) Extracellular view of the M₃ mAChR parallel to the path of the exofacial segment of TM III. As discussed in the text, muscarinic agonists promote the formation of a disulfide bond between C532^7.42 and a Cys residue introduced at position 151^3.36 [21]. (b) Predicted location of I169^3.54, K484^6.29, and A488^6.33 in the 3D structure of the inactive state of the M₃ mAChR (cytoplasmic view). K484^6.29 and A488^6.33, which are located at the cytoplasmic end of TM VI, are projecting away from I169^3.54 present at the bottom of TM III. Disulfide cross-linking data support the view that muscarinic agonists trigger a conformational change that includes a clockwise rotational movement of the cytoplasmic segment of TM VI [22]. (c) Predicted location of L249^5.57-I253^5.61 (TM V) and A489^6.34-L492^6.37 (TM VI) in the inactive state of the M₃ mAChR (cytoplasmic view). Among the five highlighted TM V residues, only Y250^5.58 and I253^5.61 are predicted to face TM VI. Disulfide scanning mutagenesis studies strongly suggest that the cytoplasmic end of TM VI, but not the corresponding region of TM V, undergoes a major activity-dependent conformational change (see text for more details; [24]). (d) Cytoplasmic view of the inactive state of the M₃ mAChR showing the locations of residues V88^1.53-F92^1.57 (TM I) and V541^7.51-C546^7.56 (TM VII). Cys substitution of the residues highlighted in yellow resulted in double Cys mutant receptors that showed agonist-dependent disulfide cross-linking (88^1.53/543^7.53, 91^1.56/545^7.55, and 91^1.56/546^7.56) [20]. Whereas V88^1.53 and Y543^7.53 directly face each other in the inactive state of the M₃ muscarinic receptor, L545^7.55 and C546^7.56 point towards the lipid face and TM VI, respectively, away from A91^1.56, indicating that the cytoplasmic end of TM VII undergoes a major activity-dependent structural change during receptor activation (see text for more details; [20]). (e) Cytoplasmic view of a selected region of the intracellular surface of the M₃ mAChR. Disulfide cross-linking data suggest that muscarinic agonists increase the distance between Cys residues introduced at a) positions 91^1.56 and 549^7.59 and b) positions 92^1.57 and 550^7.60 (these four residues are highlighted in yellow) [23]. In contrast, inverse muscarinic agonists are predicted to reduce the distance between these residues [23]. The individual figure panels were taken from Refs [21]-[24]. Numbers refer to amino acid positions in the rat M₃ mAChR sequence [33].

Figure 4

Summary of activity-dependent structural changes predicted to occur at the cytoplasmic surface of the M₃ mAChR. A 3D model of the inactive state of the rat M₃ mAChR was built via homology modeling using the high-resolution X-ray structure of the β₂-adrenergic receptor [4, 5] as a template (S. Costanzi and J. D. Karpiak; unpublished results). The intracellular surface of the M₃ mAChR is viewed from the cytoplasm. As discussed in detail in the text, disulfide cross-linking data suggest that agonist binding to the M₃ mAChR causes the following structural changes: 1) The cytoplasmic end of TM VI is predicted to undergo a rotational movement (and perhaps a partial unfolding) and to move closer to the corresponding segment of TM V [18, 22, 24]. 2) The C-terminal portion of TM VII is thought to move closer to the corresponding region of TM I, accompanied by a rotational movement (and perhaps a partial unfolding) of this TM VII segment [20]. 3) The N-terminal portion of helix 8 is predicted to move away from the cytoplasmic end of TM I [23]. The agonist-induced structural changes are thought to expose previously inaccessible receptor residues or surfaces (e.g. on the cytoplasmic ends of TM III, VI, and VII), ultimately resulting in productive receptor/G protein coupling.