Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C

K Okaichi, AB Cubitt, GS Pitt and RA Firtel

Department of Biology, University of California, San Diego, La Jolla 92093-0634.

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.

Volume 3, Issue 7, pp. 735-747, 07/01/1992
Copyright © 1992 by The American Society for Cell Biology

This article has been cited by other articles:

				B. Sun and R. A. Firtel A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development in Dictyostelium Mol. Biol. Cell, April 1, 2003; 14(4): 1727 - 1743. [Abstract] [Full Text] [PDF]

				Genetically Modified Animals in Endocrinology Endocr. Rev., April 1, 2002; 23(2): 276 - 278. [Full Text] [PDF]

				D. T. Brazill, D. F. Lindsey, J. D. Bishop, and R. H. Gomer Cell Density Sensing Mediated by a G Protein-coupled Receptor Activating Phospholipase C J. Biol. Chem., April 3, 1998; 273(14): 8161 - 8168. [Abstract] [Full Text] [PDF]

				T. Chen, W. Wolf, and R. Chisholm Cell-type-specific rescue of myosin function during Dictyostelium development defines two distinct cell movements required for culmination Development, January 10, 1998; 125(19): 3895 - 3903. [Abstract] [PDF]

				M. Maeda and R. A. Firtel Activation of the Mitogen-activated Protein Kinase ERK2 by the Chemoattractant Folic Acid in Dictyostelium J. Biol. Chem., September 19, 1997; 272(38): 23690 - 23695. [Abstract] [Full Text] [PDF]

				H Aizawa, Y Fukui, and I Yahara Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles J. Cell Sci., January 10, 1997; 110(19): 2333 - 2344. [Abstract] [PDF]

				S Lee, R Escalante, and R. Firtel A Ras GAP is essential for cytokinesis and spatial patterning in Dictyostelium Development, January 3, 1997; 124(5): 983 - 996. [Abstract] [PDF]

				M. Maeda, L. Aubry, R. Insall, C. Gaskins, P. N. Devreotes, and R. A. Firtel Seven Helix Chemoattractant Receptors Transiently Stimulate Mitogen-activated Protein Kinase in Dictyostelium J. Biol. Chem., February 16, 1996; 271(7): 3351 - 3354. [Abstract] [Full Text] [PDF]

				J. Hadwiger, K Natarajan, and R. Firtel Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis Development, January 4, 1996; 122(4): 1215 - 1224. [Abstract] [PDF]

				R A Firtel Integration of signaling information in controlling cell-fate decisions in Dictyostelium. Genes & Dev., June 15, 1995; 9(12): 1427 - 1444. [PDF]

				M. Lydan and D. Cotter The role of Ca2+ during spore germination in Dictyostelium: autoactivation is mediated by the mobilization of Ca2+ while amoebal emergence requires entry of external Ca2+ J. Cell Sci., January 5, 1995; 108(5): 1921 - 1930. [Abstract] [PDF]

				S Dharmawardhane, A. Cubitt, A. Clark, and R. Firtel Regulatory role of the G alpha 1 subunit in controlling cellular morphogenesis in Dictyostelium Development, January 12, 1994; 120(12): 3549 - 3561. [Abstract] [PDF]

				P Lilly, L Wu, D L Welker, and P N Devreotes A G-protein beta-subunit is essential for Dictyostelium development. Genes & Dev., June 1, 1993; 7(6): 986 - 995. [Abstract] [PDF]

				H Flaadt, E Jaworski, and D Malchow Evidence for two intracellular calcium pools in Dictyostelium: the cAMP-induced calcium influx is directed into a NBD-Cl- and 2,5-di-(tert-butyl)1,4-hydroquinone-sensitive pool J. Cell Sci., January 8, 1993; 105(4): 1131 - 1135. [Abstract] [PDF]

				J. Roelofs, H. M. Loovers, and P. J. M. Van Haastert GTPgamma S Regulation of a 12-Transmembrane Guanylyl Cyclase Is Retained after Mutation to an Adenylyl Cyclase J. Biol. Chem., October 26, 2001; 276(44): 40740 - 40745. [Abstract] [Full Text] [PDF]