Synergistic effects of peptide GEFs on Gα i1. These results suggest that KB-752 and D2N impart GEF activity by distinct mechanisms and that robust exchange by Gα is achieved through a multiple-site mechanism.įig. Similar to GTPγS binding, both KB-752 and D2N modestly enhanced steady-state GTP hydrolysis however, their combined effect was again synergistic. We also assessed the effects of each peptide under steady-state GTP hydrolysis conditions in which nucleotide exchange is rate-limiting ( 15). When combined, KB-752 and D2N resulted in a synergistic enhancement of the initial GTPγS binding rate ( Fig. To investigate these peptide effects more fully, we focused on initial GTPγS binding rates under each condition. Peptide effects were predominantly seen in the initial reaction rate modest effects on the overall magnitude of GTPγS binding to Gα i1 likely reflect the ability of these peptides to reduce the requirement on magnesium for GTPγS binding similar to that displayed by activated receptors ( 9, 14). Notably, combining KB-752 and D2N resulted in a rate of GTPγS binding approaching that of a full-length receptor acting on the Gαβγ heterotrimer in reconstituted systems and cell membrane preparations ( 13). When used alone (at maximally effective concentrations see refs 9 and 11), KB-752 and D2N each stimulated the rate of GTPγS binding ( Fig. D2N and several other receptor-derived peptides have been shown to elicit modest exchange factor activity in vitro toward specific Gα subunits and are therefore thought to represent direct receptor/Gα engagement sites acted on during GPCR-mediated activation ( 12). ( 9) recently described a peptide with GEF activity, D2N, derived from the N-terminal region of the third intracellular (ic3) loop of the D2-dopamine receptor. We have determined the structure of a complex of these two GEF peptides bound to Gα i1, a structure that has revealed critical determinants for receptor/Gα coupling and receptor-mediated nucleotide exchange. Because these studies with KB-752 were illustrative only of the Gβγ contribution to heterotrimer activation by activated receptor, here we investigated the effects of KB-752 on Gα in combination with a receptor fragment that also possesses GEF activity. We recently reported the structure of Gα i1 bound to a Gβγ-surrogate peptide, KB-752, which acts as a Gα GEF by displacing the occlusive β3/α2 lip in an analogous manner to that proposed for receptor-mediated tilting of Gβγ during activation ( 5, 11). Modulation of the α5 helix, in turn, is suggested to alter the β6/α5 loop conformation and destabilize contacts with the guanine ring of GDP, thus allowing for nucleotide release. An alternative hypothesis suggests that receptors use the extreme C terminus of Gα as a “latch” to alter the conformation of the α5 helix ( 6, 9, 10). The β3/α2 loop in this model serves as an occlusive barrier to GDP release, and receptor-promoted alterations in its conformation (i.e., removal from the GDP-binding pocket) thereby create a feasible egress route for GDP. One proposed model suggests that the receptor uses Gβγ as a “lever” to reorient the β3/α2 loop of Gα ( 5). One unifying aspect of several current models of GPCR GEF activity ( 4– 6) is that ligand-activated receptor must act at a distance to transmit conformational changes through distinct regions of the G protein, because the GDP-binding pocket of Gα resides ≈30 Å away from the receptor, precluding the possibility of direct manipulation of this region in its proposed orientation at the receptor interface ( 7, 8).
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