Role of Isoprenoid Lipids on the Heterotrimeric G Protein γ Subunit in Determining Effector Activation

Abstract

Post-translational prenylation of heterotrimeric G protein ␥ subunits is essential for high affinity ␣-␤␥ and ␣-␤␥-receptor interactions, suggesting that the prenyl group is an important domain in the ␤␥ dimer. To determine the role of the prenyl modification in the interaction of ␤␥ dimers with effectors, the CAAX (where A indicates alipathic amino acid) motifs in the ␥ 1 , ␥ 2 , and ␥ 11 subunits were altered to direct modification with different prenyl groups. Six recombinant ␤␥ dimers were overexpressed in baculovirus-infected Sf9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-␤ isozymes and type II adenylyl cyclase. The native ␤ 1 ␥ 2 dimer (␥ subunit modified with geranylgeranyl) is more potent and effective in activating phospholipase C-␤ than either the ␤ 1 ␥ 1 (farnesyl) or the ␤ 1 ␥ 11 (farnesyl) dimers. However, farnesyl modification of the ␥ subunit in the ␤ 1 ␥ 2 dimer (␤ 1 ␥ 2-L71S) caused a decrement in its ability to activate phospholipase C-␤. In contrast, both the ␤ 1 ␥ 1-S74L (geranylgeranyl) and the ␤ 1 ␥ 11-S73L (geranylgeranyl) dimers were more active than the native forms. The ␤ 1 ␥ 2 dimer activates type II adenylyl cyclase about 12-fold; however, neither the ␤ 1 ␥ 1 nor the ␤ 1 ␥ 11 dimers activate the enzyme. As was the case with phospholipase C-␤, the ␤ 1 ␥ 2-L71S dimer was less able to activate adenylyl cyclase than the native ␤ 1 ␥ 2 dimer. Interestingly, neither the ␤ 1 ␥ 1-S74L nor the ␤ 1 ␥ 11-S73L dimers stimulated adenylyl cyclase. The results suggest that both the amino acid sequence of the ␥ subunit and its prenyl group play a role in determining the activity of the ␤␥-effector complex.Heterotrimeric G proteins 1 are transducers of numerous extracellular signals from seven transmembrane receptors to intracellular effectors (1-4). G proteins are composed of ␣, ␤, and ␥ subunits and associate with the inner side of the plasma membrane. Receptor activation catalyzes the exchange of GDP for GTP on the ␣ subunit, resulting in dissociation of the GTP-liganded ␣ subunit from the ␤␥ dimer (1). Both the GTPbound form of the ␣ subunit and the released ␤␥ dimer regulate a variety of effectors, including PLC-␤ (5, 6) and adenylyl cyclases (7,8). To date, 7 ␤ subunits and 11 ␥ subunits have been identified in mammalian systems (9 -15). Selective assembly of ␤␥ heterodimers from these proteins may produce a large number of unique complexes that differ in their interactions with ␣ subunits, receptors, and effectors. While the first four ␤ subunits identified, ␤ 1 -␤ 4 , are 85-90% identical in primary amino acid sequence (9), the ␥ subunits are much more divergent. For example, the ␥ 1 and ␥ 5 subunits are only 25% identical. Thus, the ␥ subunits may impart some specificity to the ␤␥ signal. The G protein ␥ subunits are subject to post-translational modification by the addition of isoprenoid lipids to an invariant cysteine residue in the CAAX motif at their C terminus (15-17). The last amino acid (X) of the CAAX motif is an important determinant for...

G protein βγ complex translocation from plasma membrane to Golgi complex is influenced by receptor γ subunit interaction

“…3A). Mass spectrometry has been used to show that 90% of a γ11 mutant containing the geranylgeranylation target CAAX sequence and a γ2 mutant containing the CAAX sequence for farnesylation were appropriately and fully processed even in insect cells although these proteins were of mammalian origin [29]. Since the cells used here are mammalian, the mutant proteins are expected to be processed appropriately here and in the experiments below.…”

Differential Sensitivity of Phosphatidylinositol 3-Kinase p110γ to Isoforms of G Protein βγ Dimers

“…Verification of the proper post-translational processing of the ␥ subunit in the dimer was accomplished using matrix-assisted laser desorption ionization mass spectrometry as described (38). In keeping with our previous experience (34,38), the ␥ subunits in the dimers used in this study were fully and properly modified with the geranylgeranyl (␥ 2 , ␥ 10 , ␥ 12 , ␥ 13 ) or farnesyl (␥ 11 , ␥ 1 ) isoprenoid groups. Moreover, the ␥ subunits engineered to have altered CAAX sequences (e.g.…”

“…Construction of Recombinant Baculoviruses-The methods for constructing the G protein baculoviruses used in this study have been published as follows: (33,34). The baculovirus for ␥ 13 was the kind gift of Dr. David Siderovski, University of North Carolina (35).…”

Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

“…The dynamic equilibrium may be critical to the biochemical activity of the proteins since the interaction of the prenylated proteins with other macromolecules may be significantly affected by the methylation state of the adjacent −COOH group. This is perhaps more substantial based on the fact that the isoprenyl group of proteins such as the G-protein γ subunits are essential for their coupling of receptor-derived signals to such effector enzymes as the adenylyl cyclases and phospholipase Cβ2 [2,4,5]. Furthermore, an isoprenyl-binding site has been identified using solution NMR on Rho dissociation inhibitor [6].…”