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. 1993 Feb 1;90(3):794–798. doi: 10.1073/pnas.90.3.794

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

D E Wildman 1, H Tamir 1, E Leberer 1, J K Northup 1, M Dennis 1
PMCID: PMC45756  PMID: 8430087

Abstract

Guanine nucleotide-binding regulatory protein (G protein) beta gamma dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein beta 1 gene or a bovine G-protein gamma 2 gene. It was possible to express the beta 1 and gamma 2 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant beta and/or gamma chains to function in defined biochemical assays of beta gamma activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing beta or gamma chain alone were inactive in these assays, whereas those from cells coinfected with beta 1 and gamma 2 did display activity. These assays were used to identify recombinant beta gamma dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble beta gamma dimers facilitated rhodopsin-catalyzed guanosine 5'-[gamma-thio]triphosphate binding to Gt alpha, the GTP-binding subunit of the retinal G protein transducin (K0.5 of 13 +/- 2 and 36 +/- 5 nM, respectively). Both recombinant beta gamma dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gt alpha with equal potency (K0.5 of 9 +/- 1 and 10 +/- 3 nM for membrane and soluble dimers, respectively). [3H]Mevalonolactone labeling showed that the gamma 2 subunits of membrane-associated beta gamma dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of gamma 2 directs the membrane association of the beta 1 gamma 2 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.

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Selected References

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