Helmut Drobny

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

The majority of cancer cells derived from epithelial tissue express Lewis-Y (LeY) type difucosylated oligosaccharides on their plasma membrane. This results in the modification of cell surface receptors by the LeY antigen. We used the epidermal growth factor (EGF) receptor family members ErbB1 and ErbB2 as model systems to investigate whether the sugar moiety can be exploited to block signaling by growth factor receptors in human tumor cells (i.e., SKBR-3 and A431, derived from a breast cancer and a vulval carcinoma, respectively). The monoclonal anti-LeY antibody ABL364 and its humanized version IGN311 immunoprecipitated ErbB1 and ErbB2 from detergent lysates of A431 and SKBR-3, respectively. ABL364 and IGN311 blocked EGF- and heregulin-stimulated phosphorylation of mitogen-activated protein kinase [MAPK = extracellular signal-regulated kinase 1/2] in SKBR-3 and A431 cells. The effect was comparable in magnitude with that of trastuzumab (Herceptin) and apparently noncompetitive with respect to EGF. Stimulation of MAPK by ErbB was dynamin dependent and contingent on receptor internalization. ABL364 and IGN311 changed the intracellular localization of fluorescent EGF-containing endosomes and accelerated recycling of intracellular [(125)I]EGF to the plasma membrane. Taken together, these observations show that antibodies directed against carbohydrate side chains of ErbB receptors are capable of inhibiting ErbB-mediated signaling. The ability of these antibodies to reroute receptor trafficking provides a mechanistic explanation for their inhibitory action.