Hans Hofstetter

Proton-sensing G-protein-coupled receptors

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8—site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

Powerful and versatile enhancer-promoter unit for mammalian expression vectors

The enhancer and promoter of the immediate early gene of human cytomegalovirus (hCMV) were tested as a transcriptional control element by fusion to the cat gene, followed by measurement of its expression from plasmid DNA in the extrachromosomal and integrated state, respectively. Comparison of the hCMV enhancer-promoter to two other viral elements was performed in six commonly used cell lines of different tissue and species origin. Irrespective of the cell line and of the state of the DNA, the hCMV enhancer-promoter was considerably stronger than both the SV40 promoter and the long terminal repeat of Rous sarcoma virus.