MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation.

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Distinct contributions of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to protective immunity against influenza.

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.