LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM.

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway.

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca(2+) mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H(2)O(2) treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.

Role of the G-protein-coupled receptor GPR12 as high-affinity receptor for sphingosylphosphorylcholine and its expression and function in brain development.

Lysophospholipids are bioactive molecules influencing numerous cellular processes such as proliferation, differentiation, and motility. As extracellular ligands, they interact with specific members of the G-protein-coupled receptor family. We show in this paper that the lysophospholipid sphingosylphosphorylcholine is a high-affinity ligand for the orphan G-protein-coupled receptor GPR12. Heterologous expression of GPR12 in Chinese hamster ovary cells and in frog oocytes revealed a high-affinity interaction with sphingosylphosphorylcholine in the nanomolar range. Blockade of its action by pertussis toxin was taken as evidence that GPR12 is coupled to an inhibitory G-protein. In the adult mouse brain, GPR12 was expressed in the limbic system. During mouse embryonal development, GPR12 transcripts were detected in the CNS, especially in areas where neuronal differentiation occurs. Consistent with this we found that cultures of embryonal cerebral cortical neurons responded to sphingosylphosphorylcholine with an increase in synaptic contacts. The GPR12-expressing hippocampal cell line HT22 reacted to sphingosylphophorylcholine with an increase in cell proliferation and cell clustering. Other receptors known to interact at nanomolar concentrations with sphingosylphosphorycholine were expressed neither in the developing cerebral cortex nor in the HT22 cell line. We therefore hypothesize that sphingosylphosphorylcholine, most likely by interaction with GPR12, has positive effects on the differentiation and maturation of postmitotic neurons and that it may also influence the proliferation of neuronal precursor cells.

Characterization of the VPS10 domain of SorLA/LR11 as binding site for the neuropeptide HA.

The single transmembrane receptor sorLA/LR11 contains binding domains typical for the low-density lipoprotein receptors and a VPS10 domain which, in the related receptor sortilin, binds the neuropeptide neurotensin. SorLA is synthesized as a proreceptor which is processed to the mature form by a furin-like propeptidase. Endogenous sorLA and its hydra homologue HAB bind the neuropeptide head activator (HA). Transiently expressed partial sorLA constructs were investigated for ligand binding. We found that HA binds with nanomolar affinity to the VPS10 domain. The sorLA propeptide also bound to the VPS10 domain, whereas the receptor-associated protein RAP interacted both with the class A repeats and the VPS10 domain. The sorLA propeptide inhibited binding of HA to full-length sorLA and to the VPS10 domain. It also interfered with binding of HA to hydra HAB, which is taken as evidence for a highly conserved tertiary structure of the VPS10 domains of this receptor in hydra and mammals. The propeptide inhibited HA-stimulated mitosis and proliferation in the human neuroendocrine cell line BON and the neuronal precursor cell line NT2. We conclude that sorLA is necessary for HA signaling and function.