Received for publication September 15, 2005.
Revised November 29, 2005.
Accepted for publication November 30, 2005.

Regions in the G Protein Subunit Important for Interaction with Receptors and Effectors

Abstract

G dimers containing the ₁₁ or ₁ subunits are often less potent and effective in their ability to regulate effectors as compared to dimers containing the ₂ subunit. To explore the regions of the subunit which affect the activity of the dimer, we constructed 8 chimeric subunits based on the ₁ and ₂ subunits. Two chimeras were made in which the N-terminal regions of ₁ and ₂ were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All 8 chimeras were expressed in Sf9 cells with the ₁ subunit, G dimers purified, and assayed for their ability to bind to the G_i1 subunit, to couple G_i1 to the A1 adenosine receptor, to stimulate phospholipase C- and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ₂ subunit modified with the geranylgeranyl lipid had the highest affinity for G_i1 (range of 0.5-1.2 nM) and were most effective at coupling the G_i1 subunit to receptor. These dimers were most effective at stimulating PLC- and inhibiting type I adenyl cyclase. In contrast, dimers containing the N-terminal sequence of the ₂ subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate both the N and C terminal regions of the subunit impart specificity to receptor and effector interactions.

Key words: Adenosine, Gi family, Adenylyl cyclases, Phospholipase C's, cAMP, G protein regulation

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