Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization.

Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells.

This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).

Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.

Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells.

Cell cycle dependence of protein subcellular location inferred from static, asynchronous images.

Protein subcellular location is one of the most important determinants of protein function during cellular processes. Changes in protein behavior during the cell cycle are expected to be involved in cellular reprogramming during disease and development, and there is therefore a critical need to understand cell-cycle dependent variation in protein localization which may be related to aberrant pathway activity. With this goal, it would be useful to have an automated method that can be applied on a proteomic scale to identify candidate proteins showing cell-cycle dependent variation of location.

Developmental Expression of Echinonectin, an Endogenous Lectin of the Sea Urchin Embryo.

Echinonectin (EN) is a galactose-binding lectin present in eggs and embryos of the sea urchin Lytechinus variegatus. Recent studies have suggested that EN is a hyaline layer protein that may function as a substrate adhesion molecule (SAM) during development. We have used monoclonal and affinity-purified polyclonal antibodies that specifically recognize this protein to determine its spatial and temporal expression during embryogenesis.