Mechanism of Allosteric Regulation of the Rod cGMP Phosphodiesterase Activity by the Helical Domain of Transducin α Subunit

Abstract

The G protein ␣ subunit (G␣) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an ␣ helical domain (HD). We have recently reported that the helical domain (HD t ) of the vertebrate visual transducin ␣ subunit (G␣ t ) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated G␣ t (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878 -12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin ␣ subunits, but no other G␣ HD proteins, each attenuate the PDE catalytic core (P␣␤) and synergize G␣ t stimulation of the holoPDE (P␣␤␥ 2 ) with similar apparent affinities. The data from studies of both HD t -mediated attenuation and stimulation indicate that the HD t and the PDE inhibitory subunit (P␥) interact with PDE at independent sites and that P␣␤ contains the binding sites for HD. The saturation of both processes by HD t displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of P␣␤ activity and 2.1 for synergism of holoPDE activation. Our data suggest the that G␣ t -HD t regulates PDE by allosterically decreasing the affinity of P␣␤ for P␥ and thus simultaneously facilitating the interaction of the activated G␣ t -Ras-like domain with P␥. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.G proteins (heterotrimeric guanine nucleotide-binding proteins) play a central role in many cell-signaling processes. Among the ␣, ␤, and ␥ subunits of heterotrimeric G proteins, the ␣ subunit (G␣) has received the most attention because most of the known structural determinants for receptor-G protein or effector-G protein interactions reside in G␣ proteins (1-3). During signaling, the active GTP-bound and inactive GDP-bound G protein ␣ subunits function as conformational switches that couple the extensive superfamily of cell-surface receptors to diverse intracellular effectors, including cyclic GMP phosphodiesterases, adenylyl cyclases, receptor kinases, phospholipases, and ion channels (3). Solutions for the crystal structures of the ␣ subunits of transducin (G t ) and G i reveal that these proteins essentially fold into two separate domains termed the GTPase or Ras-like domain (RasD) 1 and an ␣ helical domain (HD) (4, 5). To date, all sites for G␣ subunit interactions with receptors and effectors have been mapped to the RasD and the N-terminal segment (2-4, 6 -8); little is known about the corresponding function(s) of the HD. The HD is unique to the heterotrimeric G proteins, whereas the RasD is present in all members of the GTPase superfamily. Comparison of the amino acid sequences reveals that diversity in the HD is remarkably greater than in the RasD among G␣ families (5, 9).2 These observations implicate the HD a...

A Switch 3 Point Mutation in the α Subunit of Transducin Yields a Unique Dominant-negative Inhibitor

“…In other cases, ␣T* was assayed in the presence of rhodopsin and G␤␥ (f) or in the absence of rhodopsin (E), and ␣T*(R238E) was assayed in the presence of rhodopsin and G␤␥ (OE). The results are representative of three experiments (31)(32)(33), it was attractive to consider that interactions between Switch 3 and the helical domain might be essential for effector regulation. Since we already had shown that changing the position of the helical domain relative to the GTPase domain can influence GDP-GTP exchange, we also wondered whether disrupting interactions between Switch 3 and the helical domain might impact G␣ activation.…”

“…1b), which is in the phosphate-binding P-loop lying just upstream from the beginning of the large helical domain, whereas in the activated (GTP␥S-bound) ␣T subunit, Arg-238 contacts both Glu-39 and Gln-143 of the helical domain. It has been suggested that the helical domain of G␣ subunits plays a role in mediating the regulation of target/effectors, and in the particular case of ␣T, may be essential for the stimulation of PDE activity (31)(32)(33). Thus, we wanted to see whether disruption of the apparent links between the conserved Arg-238 in Switch 3 and Glu-39 and/or Gln-143 might compromise ␣T activation and/or its ability to bind and regulate the PDE.…”

A regulator of G protein signaling interaction surface linked to effector specificity

“…In addition to switch III residues shown to participate in G t␣ -PDE␥ binding, class-specific residues g91, g142, g143, and g144, found in the helical domain, are exposed on the surface of G ␣ . A recent study reported the surprising result that the helical domain alone can increase the activity of PDE in a G ␣ -specific manner (47,48). However, the helical domain is almost entirely devoid of ET surface signal, with the exception of residues g91 and g142-g144.…”

Light- and Guanosine 5′-3-O-(Thio)triphosphate-sensitive Localization of a G Protein and Its Effector on Detergent-resistant Membrane Rafts in Rod Photoreceptor Outer Segments

“…PDE activity was assayed by a phosphate release assay as described previously (16). Protein assay was performed routinely by the Bradford assay (17).…”