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Rhodopsin-family receptors associate with small G proteins to activate phospholipase D

Abstract

G-protein-coupled receptors of the rhodopsin family transduce many important neural and endocrine signals. These receptors activate heterotrimeric G proteins and in many cases also cause activation of phospholipase D, an enzyme that can be controlled by the small G proteins ARF and RhoA1,2,3. Here we show that the activation of phospholipase D that is induced by many, but not all, Ca2+-mobilizing G-protein-coupled receptors is sensitive to inhibitors of ARF and of RhoA. Receptors of this type were co-immunoprecipitated with ARF or RhoA on exposure to agonists, and the effects of GTP analogues on ligand binding to the receptor changed to a profile that is characteristic of small G proteins. These receptors contain the amino-acid sequence AsnProXXTyr in their seventh transmembrane domain, whereas receptors capable of activating phospholipase D without involving ARF contain the sequence AspProXXTyr. Mutation of this latter sequence to AsnProXXTyr in the gonadotropin-releasing hormone receptor conferred sensitivity to an inhibitor of ARF, and the reciprocal mutation in the 5-HT2A receptor for 5-hydroxytryptamine reduced its sensitivity to the inhibitor. Receptors carrying the AsnProXXTyr motif thus seem to form functional complexes with ARF and RhoA.

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Figure 1: Properties of agonist-evoked PLD responses in 1321N1 cells and co-immunoprecipitation of M3 receptors with ARF1/3 and RhoA antibodies.
Figure 2: Immunoblots for ARF and Rho on immunoprecipitates generated with polyclonal ARF1/3, RhoA and receptor antibodies.
Figure 3: PLD and PLC responses of wild-type, Asn 318 and Asp 318 plus Asp 87 Asn 318 mutant GnRH receptors expressed in COS 7 cells and receptor immunoprecipitation with ARF1/3 and RhoA antibodies.

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Acknowledgements

R.M., E.L. and M.J. are members of the Membrane Biology Group, University of Edinburgh. This work was funded by the MRC and the NIH. M.F. was supported in part by Wellcome Research Laboratories. We thank M. Wakelam for the polyclonal ARF antibody; G. Bokoch for RhoA constructs; B. Wolfe for M3 receptor antiserum; the Scottish Antibody Production Unit for secondary antibodies; R. Clegg, L. Garland, N. Birdsall, B. Dickey, H. Weinstein, B. Ebersole, S. Dracheva and T.Harmar for help and advice; J. Bennie and S. Carroll for ligand iodination; and M. Eastwood for secretarial assistance.

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Correspondence to Rory Mitchell.

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Mitchell, R., McCulloch, D., Lutz, E. et al. Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature 392, 411–414 (1998). https://doi.org/10.1038/32937

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