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J Biol Chem, Vol. 274, Issue 24, 17365-17371, June 11, 1999

Ribozyme Approach Identifies a Functional Association between the G Protein ₁₇ Subunits in the -Adrenergic Receptor Signaling Pathway

Qin Wang, Bashar K. Mullah, and Janet D. Robishaw

From the Henry Hood M.D. Research Program, Pennsylvania State University College of Medicine, Danville, Pennsylvania 17822 and  Applied Biosystems, Foster City, California 94404

The complex role that the heterotrimeric G proteins play in signaling pathways has become increasingly apparent with the cloning of countless numbers of receptors, G proteins, and effectors. However, in most cases, the specific combinations of  and subunits comprising the G proteins that participate in the most common signaling pathways, such as -adrenergic regulation of adenylyl cyclase activity, are not known. The extent of this problem is evident in the fact that the identities of the subunits that combine with the  subunit of G_s are only now being elucidated almost 20 years after its initial purification. In a previous study, we described the first use of a ribozyme strategy to suppress specifically the expression of the ₇ subunit of the G proteins, thereby identifying a specific role of this protein in coupling the -adrenergic receptor to stimulation of adenylyl cyclase activity in HEK 293 cells. In the present study, we explored the potential utility of a ribozyme approach directed against the ₇ subunit to identify functional associations with a particular  and _s subunit of the G protein in this signaling pathway. Accordingly, HEK 293 cells were transfected with a ribozyme directed against the ₇ subunit, and the effects of this manipulation on levels of the  and _s subunits were determined by immunoblot analysis. Among the five  _s subunits detected in these cells, only the ₁ subunit was coordinately reduced following treatment with the ribozyme directed against the ₇ subunit, thereby demonstrating a functional association between the ₁ and ₇ subunits. The mechanism for coordinate suppression of the ₁ subunit was due to a striking change in the half-life of the ₁ monomer versus the ₁ heterodimer complexed with the ₇ subunit. Neither the 52- nor 45-kDa subunits were suppressed following treatment with the ribozyme directed against the ₇ subunit, thereby providing insights into the assembly of the G_s heterotrimer. Taken together, these data show the utility of a ribozyme approach to identify the role of not only the  subunits but also the  subunits of the G proteins in signaling pathways.

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