Current Awareness

Life on the edg

G protein-coupled receptors constitute one of the most abundant entities in cellular communication. Elucidation of their structure and function as well as of their regulation began 30–40 years ago and the advance has markedly increased during the last 15 years. They participate in a plethora of cell functions such as regulation of metabolic fluxes, contraction, secretion, differentiation, or proliferation, and in essentially all activities of our organism; these receptors are targets of a large proportion of prescribed and illegal drugs. Fluorescence techniques have been used to study receptors for many years. The experimental result was usually a two-dimensional (2D) micrograph. Today, the result can be a spatiotemporal (four-dimensional, 4D) movie. Advances in microscopy, fluorescent protein design, and computer-assisted analysis have been of great importance to increase our knowledge on receptor regulation and function and create opportunities for future research. In this review we briefly depict the state of the art of the G protein-coupled receptor field and the methodologies used to study G protein-coupled receptor location, trafficking, dimerization, and other types of receptor–protein interaction. Fluorescence techniques now permit the capture of receptor images with high resolution and, together with a variety of fluorescent dyes that color organelles (such as the plasma membrane or the nucleus) or the cytoskeleton, allow researchers to obtain a much clearer idea of what is taking place at the cellular level. These developments are changing the way we explore cell communication and signal transduction, permitting deeper understanding of the physiological and pathophysiological processes.

We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca²⁺]_i, and decreased MMP. However, those effects were only observed at concentrations higher than 10 μM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca²⁺]_i, and decreased MMP in Jurkat T cells.

Sphingosine-1-phosphate (S1P) acts on a set of G protein-coupled receptors in the plasma membrane and also as a second messenger in certain cell types. There are two possible pathways to mobilize intracellular Ca²⁺ concentration by S1P. One is through phospholipase C, and the other is through intracellular Ca²⁺ channels operated by S1P. The Mn²⁺ quenching method was applied to elucidate the action mode of S1P-induced Ca²⁺ mobilization in rat hepatocytes. In permeabilized hepatocytes, inositol trisphosphate induced Mn²⁺ quenching, and it was blocked by heparin. However, S1P did not induce Mn²⁺ quenching. Results suggest that S1P did not mobilize Ca²⁺ through intracellular Ca²⁺ channels.

The functions of lysophosphatidic acid (LPA) can be broadly divided into two classes: (1) physiological and (2) pathological roles. The role of LPA in embryonic development can be seen as early as oocyte formation. It continues in postnatal homeostasis, through its ability to impart a level of protection from both stress and local injury, by regulating cellular proliferation, apoptosis, and the reorganization of cytoskeletal fibers. LPA may function as a double-edged sword. While it helps maintain homeostasis against stress and insult, it may also augment the development and spread of pathological processes, including cancers.

We previously reported that sphingosine 1-phosphate (S1P) induces inhibition of adenylyl cyclase and activation of phospholipase C via independent G protein-coupled receptors in adult rat hepatocytes. Although S1P activation of phospholipase C and subsequent increase of intracellular Ca²⁺ concentration were enhanced during the primary culture of hepatocytes, S1P inhibition of adenylyl cyclase remained unchanged. Here, we addressed whether enhancement of S1P-induced actions is dependent on change of status from the differentiated (G₀) phase to proliferating (G₁/S) phase in hepatocytes. By employing cell-density-dependency of the transition (G₀-G₁) of hepatocytes in primary culture in vitro, it was found that the enhancement of phospholipase C activation by S1P was dependent on cell density and correlated to the G₀-G₁ transition. The correlation was further confirmed in vivo by 70% hepatectomy as a proliferating hepatocytes model. Northern blot analysis suggested an enhanced expression of S1P₂ receptor in proliferating hepatocytes.

Successful sequencing of the human genome has opened a new era in the life sciences and has greatly accelerated biomedical research. Among various research endeavors benefiting from established genomic information, one of the most fruitful areas is the research on orphan G protein-coupled receptors (GPCRs). Many intercellular mediators, including peptides, lipids, and other small molecules, have found their GPCRs in the plasma membrane, e.g., relaxin and tyramine. In the past 14 months, more than one dozen papers have been published reporting the finding of intercellular lipid mediators acting on rhodopsin family GPCRs.

This review focuses primarily on intercellular lipid mediators and their recently discovered GPCRs.