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

Abstract

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...

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

“…Within this stretch of sequence, ET analysis identifies five G ␣ -site 3 residues that are class-specific and surface-exposed, including two residues critical for G t␣ -PDE␥ binding (29), g243 and g244. Residues g306, g307, and g310 are class-specific, and were reported to be involved with PDE␥ binding (30)(31)(32)(33), although recent mutagenesis results have questioned those conclusions (34). Furthermore, residue g209 (205 in G t␣ ) is known to interact with the RGS domain and was shown to have no effect on PDE␥ binding, but mutation to alanine resulted in a substantial decrease in the ability of the mutant G t␣ to bind RGS16 (29).…”

Sequence of Interactions in Receptor-G Protein Coupling

“…Cloning, Expression, and Purification of Chimeric G t ␣/G i ␣ 1 and G i ␣ 1 Subunits-The expression vector pHis6-Gt␣*, which was generously provided by M. Natochin and N. Artemyev, contains a chimeric bovine G t ␣/rat G i ␣ 1 cDNA sequence, preceded by a nucleotide sequence encoding six histidines as an affinity tag (18). The chimeric G␣ protein contains only 16 residues from G i ␣ 1 and was shown to be similar to native G t ␣ in receptor interaction and basal nucleotide exchange.…”

“…Because of the known very low expression of the soluble transducin G␣ subunit (G t ␣) in Escherichia coli and Sf9 cells, single amino acid substitutions were introduced into G t ␣ to yield a G t ␣/G i ␣ 1 chimera that contained 16 residues of G i ␣ 1 in a G t ␣ background (18). G i ␣ 1 is not present in photoreceptor cells but belongs to the same G␣ subfamily as G t ␣ and couples to light-activated rhodopsin.…”

“…Cultures were grown at 30°C, induced with 100 M isopropyl-1-thio-␤-D-galactopyranoside at an A 600 of 0.8 and harvested 12-14 h later. Expression and purification of the G t ␣/G i ␣ 1 chimera was performed as described (18,19,22). E. coli BL21(DE3) harboring plasmids encoding G␣ subunits were induced with 100 M isopropyl-1-thio-␤-D-galactopyranoside at an A 600 of 0.8 and harvested 5-7 h later (20°C).…”

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

“…More recently, involvement of amino acid sequence 330-350 in the C-terminal region of T ␣ in binding to rhodopsin received further support by alanine scanning, chimeric receptor studies, and peptide-binding studies (9)(10)(11). Additional evidence suggested that the region around residue R310 in T ␣ also is likely to be involved (9,12,13). However, further insights into the contact sites in complex formation between light-activated rhodopsin and T are desirable.…”