SummaryPlant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DW m ) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DW m loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DW m and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DW m loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DW m changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DW m alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DW m loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.

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Expression of GTPase-deficient Gi2 a subunit (ai2) mutant polypeptides and overexpression of the wild-type oi2 polypeptide in Rat la, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat la cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient ai2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat la cells induced by expression of aj2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat la cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient ai2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat la cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.The oncogenic potential of mutant G protein ot subunit polypeptides has been implied from their presence in several neuroendocrine tumors (19,23). Mutations that inhibit the GTPase turn-off function of as, the a chain polypeptide which stimulates adenylyl cyclase, have been identified to occur with fairly high frequency in both pituitary adenomas and thyroid tumors. The role of activated as mutants in the transformation of somatotrophs and thyrocytes is somewhat predictable, because cyclic AMP (cAMP) induces a proliferative response in both cell types (7). Lyons and coworkers also found GTPase-inhibiting mutations in the Gi2 at subunit polypeptide, aOi2, in a subset of ovarian and adrenal cortical tumors (23). It is less apparent how constitutively active, GTPase-deficient ai2 mutant polypeptides could be involved in the transformation of these cell types, since no second messenger system previously characterized to be regulated by Gi2 (adenylyl cyclase inhibition, K+ channels, and PLA2) has been shown to be mitogenic in ovarian or adrenal cortical cells. However, Gi2-like proteins have been implicated in the thrombin-, bombesin-, and lysophosphatidic acid-stimulated mitogenic responses of specific cell types, including Swiss 3T3, NIH 3T3, and Rat la fibroblasts (5,21,37,42 stitutively active ai2 mutant polypeptides would be predicted to impart altered growth control when expressed in cells responding mitogenically to growth factors such as thrombin and lysophosphatidic acid. We have placed mutations that inhibit its intrinsic GTPase activity in the "i2 polypeptide, these are mutations the same as or similar to those present in the as and a2 genes found in a subgroup of neuroendocrine tumors (22, 23). When expressed in three different fibroblast cell lines (Rat la, NIH 3T3, and Swiss 3T3), the GTPasedeficient a(2 mutant polypeptides were capable of altering the growth cha...

The peptide C5a which is generated during the complement cascade is an important chemotactic factor involved in the inflammatory response. The C5a receptor (C5aR) primary sequence suggests that it has a serpentine structure of seven transmembrane domains which is typical of classical G‐protein‐coupled receptors. To investigate the signal transduction mechanism of C5a we transiently expressed the C5aR in combination with different G‐protein a subunits in human kidney 293 cells and measured the PLC activity induced upon C5a stimulation. Cotransfection of C5aR and α16 stimulated PLC while cotransfection of C5aR with either αq or α12 did not.

A B cell's response to antigen, whether it be proliferation, differentiation, anergy, or deletion, is dependent upon recognition of that antigen by the B cell antigen receptor (BCR) 1 (1-3). The receptor is a multimeric complex consisting of the antigenrecognition substructure, membrane-bound immunoglobulin non-covalently associated with heterodimer(s) of Ig-␣ and Ig-␤ (4 -6). Present evidence indicates that the cytoplasmic tails of Ig-␣ and ␤ (7) translate antigen engagement into cytoplasmic signaling events that initiate cellular responses (8 -12). Most proximally in the signaling cascade, one or more tyrosine kinases, including Syk and members of the Src family, are activated (13-15). These in turn activate a variety of pathways whose constituents include Ras, phosphatidylinositol 3-kinase, and phospholipase C (1). Embedded within the cytoplasmic tails of both Ig-␣ and ␤ is a sequence common to other multichain immune recognition receptor (MIRR) subunits including CD3␦, CD3␥, TCR, Fc␥RIII␥, and Fc⑀RI␥, termed the immunoreceptor tyrosine-based activation motif (ITAM) (16,17). The motif contains two tyrosines, both of which are critical for initiating tyrosine kinase activation (10, 18). Phosphorylation of these tyrosines facilitates the recruitment and activation of tyrosine kinases which contain SH2 domains, such as . Substrates for these kinases may also be recruited (22). The presence of the ITAM in all MIRR chains involved in signal transduction has led some to suggest that apparently heterologous chains such as CD3⑀ and TCR are functionally redundant and the presence of multiple ITAMs within each MIRR serve to increase the strength of signal which can be generated via the receptor. Evidence for this assertion has been obtained in studies of both the B and T cell antigen receptors (8,12,23,24). In contrast, we and others have provided evidence indicating that each heterologous ITAM containing chain has a distinct function (10,11,19,25,26).Many of the above studies utilized chimeras in which irrelevant extracellular and transmembrane domains were fused to the single cytoplasmic domain under study (18,27,28). Although this approach has yielded considerable insight into ITAM-containing chains, it assumes that functions observed in the isolated circumstance of a single chimera are reflective of the function of that cytoplasmic domain within the intact receptor complex. This might not be true since most ITAM-containing chains, such as Ig-␣ and Ig-␤, are expressed on cell surfaces as heterodimers (29 -32). Therefore, we postulated that within the context of a heterodimer, Ig-␣ and Ig-␤ would have new, complementary or even synergistic functions, not predicted from studies of single chain chimeras.As demonstrated in this report, Ig-␣ and ␤ have new and unpredicted functions in the context of a heterodimer. Using a novel chimera system, which allowed us to form either heteroor homodimers of the cytoplasmic domains of Ig-␣ and Ig-␤, we observed that when Ig-␤ is ligated independently it is able to activate tyrosine...