Signal Transduction in Dictyostelium fgd A Mutants with a Defective Interaction between Surface cAMP Receptors and a GTP-binding Regulatory Protein

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP~S and GDPI3S on cAMP binding are reduced; the stimulatory effect of cAMP on GTPTS binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum. T HE cellular slime mold Dictyostelium discoideum is a suitable organism to study signal transduction. Exogenous cAMP induces several responses, which lead to cell aggregation and finally to the formation of a fruiting body with spore and stalk cells (11). Stimulation of aggregative cells with cAMP induces the activation of guanylate and adenylate cyclase, leading to a rise of intracellular guanosine 3':5'-monophosphate (cGMP) t and cAMP. The increase in cGMP is transient, reaching a maximum ~10 s after stimulation. Whereas cGMP remains intracellular and is probably involved in the chemotactic reaction, the cAMP produced is secreted and the signal is thus relayed (for reviews see references 2, 5, 6). Extracellular cAMP is detected by specific cell surface receptors, which have been subdivided in two classes, Aand B-sites, that are probably coupled to the activation of adenylate and guanylate cyclase, respectively (7, 17). Binding of cAMP to both subclasses is complex and reveals in each class different forms that interconvert after stimulation of cells with cAMP (19, 20). The A-sites are fast dissociating (t,/2 = 2 s) and may exist in two states (A r~ and A L) with high and low affinity, while the B-sites are slow dissociating and may exist in at least two states (B s and B ss) which release bound cAMP with t,/, = 15 and 150 s, respectively. 1. Abbreviations used in this paper: ATP'yS, adenosine 5'-0-(3-thio)triphosphate; cGMP, guanosine 3':5'-monophosphate; dcAMP, 2'-deoxyadenosine 3':5'-monophosphate; GDPI3S, guanosine 5'-0-(2-thio)diphosphate; GTPyS, guanosine 5'-0-(3-thio)triphosphate; PB, phosphate buffer; (Sp)cAMPS, adenosine 3':5'-monophosphorothioate, Sp-isomer. Several observations point to a role of G-proteins in transmembrane signal transduction in D. discoideum. Guanine nucleotides alter the heterogeneity of cAMP binding to isolated membranes, suggesting the interaction of G-protein(s) with cAMP receptors (16, 20). [3H]GTP or [35S]GTP3,Sbinding to D. discoideum membranes and its potentiation by cAMP also point to a functional coupling between cell surface receptors and G-proteins (3, 13). Furthermore, depending on the assay conditions, GTP stimulates or inhibits adenylate cyclase activity in vitro, which supports the idea of the involvement of G-proteins (14, 21). The small haploid genome of D. discoideum makes this eukaryotic organism an excellent object to study signal transduction in chemosensory mutants. Potentially, this may provide tools to elucidate the intricacies of signal transduction pathways that are not easily obtained with other means. Mutant studies may also lead to the identification of components whose participation in signal transduction is presently unknown. We have started to analyze signal transduction in mutants which possess cAMP receptors, but do not react to cAMP with the normal set of responses. Amebas of the so-called "frigid" mutants are nearly completely unresponsive to cAMP signals (1). 11 frigid mutants were isolated; genetic evidence indicates that they belong to five complementation groups (fgd A-fgd E). Biochemical data allowed the distinction of two subtypes. One type is unable to respond to cAMP signals because development is blocked very early after starvation and so the cells do not make cell surface cAMP receptors (fgd B, D, and E). The © The Rockefeller University Press, 0021-9525/88/08/521/8 $2.00 The Journal of Cell Biology, Volume 107, August 1988 521-528 521 on July 0, 2017 jcb.rress.org D ow nladed fom other type (fgd A and C) enters the developmental pathway to some extent and does make significant levels of surface cAMP receptors. Some of these mutants show a weak chemotactic response to cAMP, but in none could differentiation be accelerated by the addition of cAMP pulses (1), These characteristics make thefgd A and C mutants very suitable for use in investigation of the cAMP signal-response coupling process. In this paper we describe the biochemical characterization of mutants from thefgd A group. We tried to determine why these cells are unable to respond to cAMP signals and where the defect in the signal transduction pathway is localized. The results show a dramatic defect in the interaction between cAMP receptors and a putative G-protein, probably caused by a defect at one of the G-proteins. Materials and Methods