Region determining specificity of G protein binding (compare chimeras 1 and 2)

activate the transducing Ga subunit. Specific regions within the C3 loop are thought to assume a unique three-dimensional structure in all receptors that bind the same G protein (e.g., Gs or Gi). Other evidence indicates that the C2 loop, joining helices 3 and 4, also contributes to the interaction of some receptors with a G protein and that residues in at least four transmembrane helices participate in ligand binding.

X-ray crystallographic analysis has pinpointed the regions in Gsa-GTP that interact with adenylyl cyclase. This enzyme is a multipass transmembrane protein with two large cytosolic segments containing the catalytic domains (Figure 13-14a). Because such transmembrane proteins are notoriously difficult to crystallize, scientists prepared two protein fragments encompassing the catalytic domains of adenylyl cyclase and allowed them to associate in the presence of Gsa-GTP and forskolin, which stabilizes the catalytic adenylyl cyclase fragments in their active conformations. The complex that formed was catalytically active and showed pharmacological and biochemical properties similar to those of intact full-length adenylyl cyclase. In this complex, two regions of Gsa-GTP, the switch II helix and the a3-p5 loop, contact the adenylyl cyclase fragments (Figure 13-14b). Recall that switch II is one of the segments of a G protein whose conformation is different in the GTP-bound and GDP-bound states (see Figure 13-8). The GTP-induced conformation of Gsa that favors its dissociation from Gp7 is precisely the conformation essential for binding of Gsa to adenylyl cyclase. Other studies indicate that Gia binds to a different region of adenylyl cyclase, accounting for its different effect on the effector.

To understand how binding of Gsa-GTP promotes adeny-lyl cyclase activity, scientists will first have to solve the structure of the adenylyl cyclase catalytic domains in their unactivated conformations (i.e., in the absence of bound Gsa-GTP). One hypothesis is that binding of the switch II helix to a cleft in one catalytic domain of adenylyl cyclase leads to rotation of the other catalytic domain. This rotation is proposed to lead to a stabilization of the transition state, thereby stimulating catalytic activity.

M EXPERIMENTAL FIGURE 13-13 Studies with chimeric adrenergic receptors identify the long C3 loop as critical to interaction with G proteins. Xenopus oocytes were microinjected with mRNA encoding a wild-type a2-adrenergic, p2-adrenergic, or chimeric a-p receptor. Although Xenopus oocytes do not normally express adrenergic receptors, they do express G proteins that can couple to the foreign receptors expressed on the surface of microinjected oocytes. The adenylyl cyclase activity of the injected cells in the presence of epinephrine agonists was determined and indicated whether the adrenergic receptor bound to the stimulatory (Gs) or inhibitory (Gj) type of oocyte G protein. Comparison of chimeric receptor 1, which interacts with Gs, and chimeric receptor 2, which interacts with Gi, shows that the G protein specificity is determined primarily by the source of the cytosol-facing C3 loop (yellow) between a helices 5 and 6. [See B. Kobilka et al., 1988, Science 240:1310.]


Adenylyl cyclase


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