Prenyl modification of guanine nucleotide regulatory protein gamma 2 subunits is not required for interaction with the transducin alpha subunit or rhodopsin.

Abstract

Guanine nucleotide-binding regulatory protein (G protein) by dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf`9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein .31 gene or a bovine G-protein 72 gene. It was possible to express the .i1 and 72 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant fi and/or y chains to function in dermed biochemical assays of fy activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing fi or y chain alone were inactive in these assays, whereas those from cells coinfected with fil and 72 did display activity. These assays were used to identify recombinant By dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble By dimers facilitated rhodopsin-catalyzed guanosine 5'-[ythioltriphosphate binding to Gta, the GTP-binding subunit of the retinal G protein transducin (KO.5 of 13 ± 2 and 36 ± 5 nM, respectively). Both recombinant fBy dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gta with equal potency (Ko.5 of 9 ± 1 and 10 ± 3 nM for membrane and soluble dimers, respectively). [3HIMevalono-lactone labeling showed that the 72 subunits of membraneassociated fBy dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of 72 directs the membrane association of the fi172 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.Guanine nucleotide-binding regulatory proteins (G proteins) are a family of proteins that serve as vital links in the chain of proteins that transduce extracellular signals to diverse intracellular effectors, such as adenylyl cyclases, phosphodiesterases, and phospholipases, and to ion channels in the membrane (1-3). All signal-transducing G proteins identified to date are membrane-associated heterotrimers composed of subunits referred to as a, 3, and 'y. The (3 and y subunits of G proteins do not dissociate under physiological conditions and thus behave essentially as a monomer; in addition, fBy dimers occur in almost all tissues as a complex mixture oftwo 3proteins (4), each bound to as many as three y subunits (5, 6). Gf3'y displays a number of in vitro activities related to the guanine nucleotide binding and activation of a subunits, including reversal of aluminum fluoride-activated Gsa (ref. 7; G., stimulatory G protein), inhibition or reversal ofguanosine 5'-[y-thio]triphosphate (GTP[yS]) binding to Ga (8,9), and enhancement of pertussis and cholera toxin catalyzed ADPribosylation of Ga subunits (10, 11). Gfy is required for efficient coupling of Ga subunits to cell surface receptors (12-15). Fina...

Stimulation of phospholipase C‐β2 by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system

“…heterotrimer [31, 461. The latter findings are, however, at odds with a recent report indicating that isoprenylation of the y2 subunit is not required for the interaction of the ply2 dimer with the a subunit of retinal transducin [32]. Most interestingly, partially purified plyzC68S, a mutant plyz dimer that corresponds to the p1y1C71S dimer used in this study, failed to inhibit calmodulin-stimulated type-I adenylyl cyclase activity and did not potentiate the stimulation of type-I1 adenylyl cyclase by activated a, [31].…”

Gβγ Affinity for Bovine Rhodopsin Is Determined by the Carboxyl-terminal Sequences of the γ Subunit

“…We confirmed that the apparent affinity for rhodopsin was Ͼ12 times higher for ␤ 1 ␥ 2 than for ␤ 1 ␥ 1 for the wild-type structures, whereas the two ␤ 1 ␥ dimers displayed no difference in affinity for retinal G␣ t . Although one study using rhodopsin to catalyze GDP/GTP exchange on the G␣ t subunit showed that the ␤ 1 ␥ 1 dimer supported a faster rate of exchange than did ␤ 1 ␥ 2 or ␤ 1 ␥ 3 (38), others have found a much lower apparent affinity for ␤ 1 ␥ 1 than for other ␤␥ dimers (20,27,30,39). The direct measurement of rhodopsin-G protein interactions using surface plasmon resonance also revealed a higher affinity of ␤ 1 ␥ 2 for rhodopsin compared with ␤ 1 ␥ 1 and showed that this difference was due to a lower dissociation rate for ␤ 1 ␥ 2 (22).…”

“…There is also ample evidence suggesting the involvement of the prenyl groups in protein-protein interactions (26 -29). Two ␤␥ dimers, ␤ 1 ␥ 1 and ␤ 1 ␥ 2 , display a Ͼ10-fold difference in their affinity for bovine rhodopsin (20,30), but no apparent difference in interaction with retinal G␣ t . Since these two ␥ subunits differ in both primary amino acid sequence and isoprenoid modification, we designed this study to examine the quantitative contributions of isoprenoid modification and protein structure to the ␤␥ dimer affinity for rhodopsin.…”

The Bombesin Receptor Subtypes Have Distinct G Protein Specificities

“…Other receptor-G protein coupling studies also supported the notion that receptors can have different affinities for ␤␥ dimers. Studies of bovine rhodopsin activation of ␣ t have found differences in apparent affinity among tissue-derived ␤␥ dimers of defined compositions or recombinant ␤␥ dimers, whereas ␣ t shows essentially no preference for ␤␥ dimers (31)(32)37). The fact that both GRP-R and NMB-R preferred bovine brain ␤␥ over ␤ 1 ␥ 2 and the diverse composition of bovine brain ␤␥ dimers (37,38) suggest there may be other ␤␥ dimer(s) having higher affinity or/and efficacy than ␤ 1 ␥ 2 in enhancing the catalytic activity of GRP-R and NMB-R on G␣ q .…”