The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N-and C-terminal halves of the ATPdependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI-and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-speci®c transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.

The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 catalyzes the uptake and phosphorylation of carbohydrates (1). It is further involved in signal transduction (2), e.g. catabolite repression (3), chemotaxis (4), and allosteric regulation of metabolic enzymes and transporters in response to the availability of carbohydrates (5, 6). The PTS is widely distributed in eubacteria and absent from eukaryotes. It generally consists of two central phosphoryl carriers, EI and HPr, and depending on the species, between 1 and 20 peripheral phosphotransferases, the integral membrane components that recognize and transport hexoses, hexitols, and disaccharides. EI and HPr are the proteins at the top of this divergent phosphorylation cascade (7). EI is a 64-kDa two-domain protein (8, 9). In the aminoterminal domain (EIN, residues 1-250) is located His-189, the amino acid transiently phosphorylated by PEP. The threedimensional structure of EIN has been elucidated (10, 11) and its mode of interaction with HPr characterized by NMR spectroscopy (12, 13). It is composed of a HPr binding ␣-helical subdomain and an ␣/␤ subdomain, structurally similar to the phosphohistidine swivel domain of pyruvate phosphate dikinase (PPDK). Phospho-EIN transfers the phosphoryl group to HPr, but it requires the presence of the carboxyl-terminal domain (EIC) to become phosphorylated by PEP (8,14). EIC contains the PEP binding site (15) and plays a crucial role in dimerization (16,17). It also confers species and subunit specificity during the phosphoryl transfer to the next phosphocarrier protein (14). EIC is proteolytically unstable and flexible. Its structure is not yet known. However, the amino acid sequence similarity between EIC and the PEP binding domain of PPDK of Clostridium symbiosum points toward similar fold and structure. There is also sequence similarity with PEP synthase (18).The mode of action of EI and the way its activity is controlled are not yet fully understood. The protein dimerizes in a temperature-dependent manner (19). The dissociation constant has been reported to shift from 20 M at 6°C to 0.9 M at 30°C. The presence of divalent cations (Mg 2ϩ or Mn 2ϩ ) and PEP also affects the equilibrium and the association and dissociation rate constants (20,21). The association rate constant is 2-3 orders of magnitude slower than in other dimeric proteins, * This work was supported by Grant 31-45838.95 from the Swiss National Science Foundation and a fellowship from the Secretaría de Estado de Educación y Universidades, Spain (to L. F. G.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed. Tel.: 41-0-31-631-3792; Fax: 41-0-31-631-4887; E-mail:garcia@ibc.unibe.ch.1 The abbreviations used are: PTS, phosphoenolpyruvate:glucose phosphotransferase system; Z-and E-Cl-PEP, (Z)-3-and (E)-3-chlorophos...

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The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IIC Man ⅐IID Man complex and the cytoplasmic IIAB Man subunit. IIAB Man has two domains (IIA and IIB) that are linked by a 60-Å long alanine-proline-rich linker. IIAB Man transfers phosphoryl groups from the phospho-histidine-containing phospho-carrier protein of the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the 6-OH of the transported hexose. IIAB Man occurs as a stable homodimer. The subunit contact is mediated by a swap of ␤-strands and an extensive contact area between the IIA domains. The H10C and H175C single and the H10C/H175C double mutants were used to characterize the phosphoryl transfer between IIA to IIB. Subunits do not exchange between dimers under physiological conditions, but slow phosphoryl transfer can take place between subunits from different dimers. Heterodimers of different subunits were produced in vitro by GuHCl-induced unfolding and refolding of mixtures of two different homodimers. With respect to wild-type homodimers, the heterodimers have the following activities: wild-type⅐H10C, 50%; wild-type⅐H175C 45%; H10C⅐H175C, 37%; and wild-type⅐H10C/H175C (double mutant), 29%. Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIAB Man . A phosphoryl group on a IIA domain can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity to 70 -80% of the control.The carbohydrate transporters of the bacterial phosphotransferase system (enzymes II of the PTS) 1 mediate uptake concomitant with phosphorylation of hexoses and hexitols.They consist of four functional units termed IIA, IIB, IIC, and IID that occur either as individual subunits in a protein complex or as independently folding domains of a multidomain protein. IIA and IIB sequentially transfer a phosphoryl group from the phosphoryl carrier protein HPr to the transported substrate. IIC and IID span the membrane and mediate substrate translocation. Substrate translocation is activated by the phosphorylation/dephosphorylation cycle of IIB (1-4). IIA and IIB of certain transporters have regulatory activity in addition to their "energy-transducing" function. For instance, IIA Glc of Escherichia coli, the gene product of crr, modulates the activities of adenylate cyclase (5, 6), glycerol kinase (7), and of the membrane permeases for lactose and maltose (8 -12). The IIB domains of some PTS transporters regulate the activity of antiterminator and transcription activator proteins (13). In the absence of the cognate substrate, the IIB domain of the ␤-glucoside transporter (IIBCA Bgl ) phosphorylates the antiterminator protein BglG and thereby inactivates it. This way, IIBCA Bgl feedbac...

Human erythroid progenitor cells were isolated from peripheral blood of healthy donors and amplified in a suspension culture system using recombinant growth factors (stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin) as well as conditioned medium from a human bone marrow stroma cell line to support cell proliferation. After 6-8 days of culture, the cell population consisted mainly of erythroid colony-forming cells (burst-forming units, BFU-Es and colony-forming units, CFU-Es). In these cells, we studied ligand-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and cAMP formation as the primary effector systems of guanine nucleotide-binding protein (G protein)-coupled receptors. The results confirmed the functional expression of receptors for adenosine (type A2B), prostaglandin E1 and isoprenaline (beta-adrenoceptor), all of which stimulated adenylyl cyclase, as well as for ADP (purinoceptor types P2T and P2U), platelet-activating factor and thrombin all of which caused a transient increase in [Ca2+]i. The efficacy of adenosine and prostaglandin E1 in stimulating cAMP formation was more than 5 times higher than that of isoprenaline, suggesting a low beta-adrenoceptor density. The response to adenosine and isoprenaline decreased by 80 and 55% respectively during maturation into the proerythroblast stage. Similarly, thapsigargin-sensitive intracellular Ca2+ stores and ligand-induced Ca2+ release declined by about 60% during the CFU-E-to-erythroblast transition. The overall functional expression pattern of G protein-coupled receptors differed from that in human erythroleukaemia cell lines or from that in platelets. Primary culture systems for nontransformed cells, such as the one presented here, thus will be indispensable for the study of the functional role of G protein-dependent signalling during haematopoiesis.