Biochemical analysis of the transducin-phosphodiesterase interaction

The G protein ␣ subunits, ␣ s and ␣ i2 , have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three ␣ subunit regions (Switches I-III). ␣ s residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of ␣ i2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of ␣ i2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both ␣ i2 and ␣ s to modulate adenylyl cyclase activity but do not affect ␣ subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of ␣ i2 in which substitutions reduce the ability of ␣ i2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of ␣ s . Thus, both ␣ i2 and ␣ s interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the ␣ subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.Upon activation by cell surface receptors, heterotrimeric G proteins transmit signals to effector proteins that regulate a wide variety of cellular processes (1-4). Receptors activate G proteins by catalyzing the replacement of GDP bound to the ␣ subunit with GTP, resulting in dissociation of ␣⅐GTP from the ␤␥ subunits. The GTPase activity of the ␣ subunit regulates the timing of deactivation and reassociation of the G protein subunits. The fidelity of cellular signaling requires that ␣ subunits modulate effector proteins only when bound to GTP and that only the appropriate ␣ subunit-effector pairs interact. GTP-dependent effector interaction most likely involves one or more of the three ␣ subunit regions that change conformation during the GTPase cycle (Switches I-III), identified by comparison of the x-ray crystal structures of the GTP␥S-bound 1 (active) and GDP-bound (inactive) forms of ␣ t (5, 6) and ␣ i1 (7,8). Differences in the amino acid sequences of the structurally conserved ␣ subunits (40% identity at the amino acid level, with 60 -90% identity within subfamilies) determine the specificity and nature of their interactions with effector proteins (9). However, the relationship between the molecular determinants of effector specificity and of GTP-dependent effector regulation is poorly understood.Regulation of adenylyl cyclase by the G protein ␣ subunits, ␣ s and ␣ i , raises issues specific for this ␣ subunit-effector interaction. ␣ s and ␣ i , which are relatively poorly conserved among the family of ␣ subunits (ϳ40% identical amino acids), both bind to adenylyl cyclase but have opposite effects on activity. Inhibiti...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

Identification of Common and Distinct Residues Involved in the Interaction of αi2 and αs with Adenylyl Cyclase

Grishina

Berlot

1997

“…Sites of chemical cross-linking of the P␥-subunit to G t ␣ were localized to within the ␣4-␤6 loop (14,15). A study using substituted peptides identified five nonconserved effector residues within this region (16). Despite the large body of evidence, the significance of the G t ␣ ␣4-␤6 region in the effector interaction remains unclear.…”

mentioning

confidence: 99%

“…Several mutants with Ala substitutions of residues from the ␣4/␤6 loop had impaired binding to R* and reduced degrees of activation (11). Interestingly, this R* binding site overlaps with a region of G t ␣, G t ␣-293-314, that has been implicated in the transducin-effector interaction (12)(13)(14)(15)(16). A synthetic peptide, G t ␣-293-314, corresponding to the ␣4-␤6 region was shown to activate PDE in vitro and to bind to P␥ (12,13).…”

mentioning

confidence: 99%

Roles of the Transducin α-Subunit α4-Helix/α4-β6 Loop in the Receptor and Effector Interactions

Natochin¹,

Granovsky

Muradov

et al. 1999

The visual GTP-binding protein, transducin, couples light-activated rhodopsin (R*) with the effector enzyme, cGMP phosphodiesterase in vertebrate photoreceptor cells. The region corresponding to the ␣4-helix and ␣4-␤6 loop of the transducin ␣-subunit (G t ␣) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the ␣4-␤6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the ␣4-␤6 loop in the R*-G t ␣ interface and suggests that the G t ␣ residues Arg 310 and Asp 311are involved in the interaction with R*. These residues are likely to contribute to the specificity of the R* recognition. Upon transduction of the visual signal in vertebrate photoreceptor cells, photoexcited rhodopsin (R*) 1 binds the retinal G protein, transducin (G t ), leading to G t activation. The ␣-subunit of G t (G t ␣) complexed with GTP is then released to stimulate the effector enzyme, cGMP phosphodiesterase (PDE), by reversing the inhibiton imposed by two PDE ␥ subunits (P␥) on the PDE catalytic dimer (P␣␤). Activated PDE rapidly hydrolyzes cGMP resulting in closure of cGMP-gated channels in the photoreceptor plasma membrane (1-3).The two central interactions of G t ␣ with R* and P␥ during visual excitation have been extensively investigated, and the G t ␣ interaction sites have been localized. Evidence points to the C terminus of G t ␣ as the major R* contact site that is critical for G t ␣ activation (4 -9). A second essential site of G t ␣ interaction with R* includes the ␣4/␤6 loop (residues 305-315) (6, 10, 11). A peptide, G t ␣-311-328, competed for the G t -R* interaction (6). The tryptic cleavage site at Arg 310 of G t ␣ was protected upon G t ␣␤␥ binding to R* (10). Several mutants with Ala substitutions of residues from the ␣4/␤6 loop had impaired binding to R* and reduced degrees of activation (11). Interestingly, this R* binding site overlaps with a region of G t ␣, G t ␣-293-314, that has been implicated in the transducin-effector interaction (12-16). A synthetic peptide, G t ␣-293-314, corresponding to the ␣4-␤6 region was shown to activate PDE in vitro and to bind to P␥ (12, 13). Sites of chemical cross-linking of the P␥-subunit to G t ␣ were localized to within the ␣4-␤6 loop (14, 15). A study using substituted peptides identified five nonconserved effector residues within this region (16). Despite the large body of evidence, the significance of the G t ␣ ␣4-␤6 region in the effector interaction remains unclear. An insertion of the G t ␣-295-314 segment into G i ␣ 1 only marginally improved the latter's ability to bind P␥ (17). This finding suggests that if the ␣4-␤6 region is important for the interaction with PDE, then likely the conserved residues within ␣4-␤6 are essential for the function of the effector. Alternatively, even small differences in G t ␣ and G i ␣ folding may interfere with the ability of G t ␣-293-314 to assume the proper effector-binding conformation in the context of G i...

“…The latter contacts have been localized to the switch II region, ␣ 3 -␤ 5 loop and ␣ 4 -␤ 6 loop of the RasD (42-44, 48). Of these, the ␣ 4 -␤ 6 loop is thus far the only region postulated to be involved in the selectivity for PDE stimulation based upon peptide saturations (46). In this study, we identify a new regulatory interface between the HD t and P␣␤ for which we propose a novel mechanism for G␣-effector regulation.…”

Section: Discussionmentioning

confidence: 89%

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

Liu

Clark

Sharma

et al. 1998

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...