cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development.

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

Many G-protein coupled receptors display a rapid decrease in ligand binding following pretreatment with agonist, cAR1, a cAMP receptor expressed early in the developmental program of Dictyostelium, mediates chemotaxis, activation of adenylyl cyclase, and gene expression changes that bring about the aggregation of 10(5) amoebae to form a multicellular structure. Occupancy of cAR1 by cAMP initiates multiple desensitization processes, one of which is an apparent reduction in binding sites. In transformed cells expressing cAR1 constitutively, Scatchard analyses revealed that this apparent loss of ligand binding is largely due to a significant reduction in the affinity of cAR1 for cAMP, A parallel increase in the dose dependence of cAR1-mediated cAMP uptake was observed, Consistent with these findings, proteolysis of intact cells and immunofluorescence suggested that cAR1 remains on the cell-surface following cAMP treatment, Finally, agonist-induced loss of ligand binding is impaired in cAR1 mutants lacking a cluster of cytoplasmic serine residues, which are targets of cAMP-induced phosphorylation.

The parallel agonist-induced phosphorylation, alteration in electrophoretic mobility, and loss of ligand binding of a guanine nucleotide-binding regulatory protein (G protein)-coupled chemoattractant receptor from Dictyostelium (cAR1) depend upon a cluster of five C-terminal domain serine residues (Caterina, M. J., Hereld, D., and Devreotes, P. N. (1995) J. Biol. Chem. 270, 4418-4423). Analysis of mutants lacking combinations of these serines revealed that either Ser(303) or Ser(304) is required; mutants lacking both serines are defective in all of these responses. Interestingly, several mutants, in eluding those substituted at only Ser(299), Ser(302), or Ser(303) or at non-serine positions within the third cytoplasmic loop, displayed an unstable mobility shift; the alteration was rapidly reversed upon cAMP removal. These mutants also exhibited subnormal extents of loss of ligand binding, which is assessed after removal of the ligand.For the wild-type receptor, we found that the stability of phosphorylation depends upon the concentration and duration of agonist pretreatment. This suggests that, following phosphorylation of Ser(303) or Ser(304), cAR1 undergoes a further transition (EC(50) approximate to 140 nM, t(1/2) approximate to 4 min) to a relatively phosphatase resistant state. We used this insight to show that, under all conditions tested, the extent of loss of binding is correlated with the fraction of cAR1 in the altered mobility form. We discuss possible relationships between cAR1 phosphorylation and loss of ligand binding.

When Dictyostelium cells are stimulated with cyclic adenosine 3',5'-monophosphate (cAMP), the major surface cAMP receptor expressed in early development, cAR1, undergoes a rapid phosphorylation and parallel decrease in electrophoretic mobility which may serve to regulate the activity of this G protein-coupled receptor. Biochemical analyses indicate the electrophoretic mobility shift is caused by phosphorylation of serine residues within the C-terminal cytoplasmic domain. The 18 serines of this domain are grouped in four clusters, designated 1 to 4 (in N- to C-terminal order). Two approaches were taken to determine the distribution of phosphorylation sites among the serine clusters. First, a proteolytic analysis of the C-terminal domain was performed. Second, mutants lacking various combinations of the serine clusters were created by site-directed mutagenesis and their abilities to undergo ligand-induced modification were determined. Both approaches yielded corroborative results consistent with the following model: the stimulus induces the addition of approximately two phosphates to cluster 1 and one to cluster 2; basal phosphorylation occurs predominantly in cluster 3 and to a lesser extent in cluster 2; and cluster 4 is not phosphorylated. The phosphorylation-deficient receptor mutants should be useful for establishing the role of ligand-induced phosphorylation of cAR1 in chemotaxis, cell-cell signaling, and gene expression.

Dictyostelium is a simple model organism for developmental studies. Under conditions of growth, single amoebae comprise a homogeneous population of cells, but when deprived of nutrients, cells aggregate to form a multicellular structure consisting of several different cell types (Fig. 1). Development is first detected when amoebae migrate to central points within a monolayer and form aggregates of many thousands of cells. Cells become tightly adherent within the aggregate to create a mound, upon which a single tip forms. This structure rises, falls over, and forms a migrating pseudoplasmodium or “slug” with the tip forming the anterior end. Slugs are about a millimeter in size and crawl about the surface. This migratory phase, which can be bypassed, is influenced by multiple factors, such as light, temperature, and pH. Culmination results in a sorocarp or fruiting body, which consists of a stalk of dead vacuolated cells...

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.

Previous studies have determined that various Qa2 serologic determinants can be removed from the surface of spleen cells by treatment with a phospholipase C. Our studies have determined that the class I molecule Qa2, expressed on the surface of spleen cells and activated T cells, behaves as an integral membrane protein based on its ability to associate with detergent micelles. Studies utilizing two purified phospholipase C have revealed that although most (90 to 95%) of the Qa2 molecules expressed on the surface of resting spleen cells are released as intact 40-kDa polypeptides associated with beta 2-microglobulin, activated T cells contain a major cell subpopulation expressing lipase-resistant Qa2 molecules. Flow cytometric analysis revealed that L3T4+-activated T cells expressed lipase-sensitive Qa2 molecules, whereas Lyt-2+ cells express lipase-resistant forms of the Qa2 molecule. The relationship between the secreted form of the Qa2 molecule and the lipase-generated soluble Qa2 molecule was investigated. Based on SDS-PAGE analysis, the secreted Qa2 molecules has a Mr of 39 kDa whereas the cell surface form released from either resting spleen or activated T cells by phosphatidylinositol-specific phospholipase C has a Mr of approximately equal to 40 kDa. Furthermore, the secreted Qa2 molecule lacks an epitope, cross-reacting determinant, often present on lipase-solubilized cell surface molecules. Thus, based on serologic and biochemical criteria, the soluble Qa2 molecules generated by an exogenous phospholipase C and the secreted Qa2 molecule are structurally distinct.

VSG lipase of Trypanosoma brucei specifically cleaves the glycosyl-phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein (VSG), releasing this protein from the plasma membrane. It also cleaves similar membrane anchors on some mammalian proteins. VSG lipase may play a role in processes such as parasite differentiation or antigenic variation. We describe here the cloning and sequencing of a cDNA encoding VSG lipase from T. brucei.

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.

The surface coat of Trypanosoma brucei is composed of many copies of the Variant Surface Glycoprotein (VSG). This protein is tethered to the cell membrane by a glycolipid moiety which contains dimyristylphosphatidylinositol. Following cell lysis, an endogenous, membrane-bound phospholipase C cleaves the glycolipid and releases the VSG in soluble form. The authors have purified a lipase which they believe is responsible for VSG release. This enzyme, designated VSG lipase, is assayed by measuring release of butanol-soluble /sup 3/H from VSG labeled with (/sup 3/H)myristate. The purification involves detergent extraction of trypanosome membranes, ammonium sulfate fractionation, hydrophobic chromatography, and cation exchange chromatography. The enzyme is purified roughly 2500 fold and is nearly homogeneous. Based on SDS-PAGE, it has an apparent subunit molecular weight of 37,000 daltons. This polypeptide co-fractionates with the activity during several fractionation procedures. The enzyme has an apparent s/sub 20,w/ of 3.8 S. The purified VSG lipase is active in the presence of EDTA; its activity is inhibited by organomercurials and stimulated by dithiothreitol. The purified enzyme releases dimyristylglycerol from VSG.

The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.

The variant surface glycoprotein of the parasite Trypanosoma brucei contains a glycolipid of unknown structure covalently attached to its COOH terminus. We have shown, by using metabolic labeling with [35S]methionine or [3H]myristic acid, precipitation with specific antibodies, and NaDodSO4/polyacrylamide gel electrophoresis, that this glycolipid is attached to the variant surface glycoprotein polypeptide within 1 min after its translation.