Classical ligands bind to the extracellular surface of their cognate receptors and activate signaling pathways without crossing the plasma membrane barrier. We selectively targeted the intracellular receptor-G protein interface by using cell-penetrating membranetethered peptides. Attachment of a palmitate group to peptides derived from the third intracellular loop of protease-activated receptors-1 and -2 and melanocortin-4 receptors yields agonists and/or antagonists of receptor-G protein signaling. These lipidated peptides-which we have termed pepducins-require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor third intracellular loop but instead require the C-tail of the receptor. Construction of such peptide-lipid conjugates constitutes a new molecular strategy for the development of therapeutics targeted to the receptor-effector interface. G protein-coupled receptors (GPCRs) play a vital role in the signaling processes that control cellular metabolism, cell growth, and motility, inflammation, neuronal signaling, and blood coagulation. Although remarkably diverse in sequence and function, all GPCRs share a highly conserved topological arrangement of a seven-transmembrane helical core domain joined by three intracellular loops, three extracellular loops, and N-and C-terminal domains (1). A key event for the switch from inactive to active receptor is ligand-induced conformational changes of transmembrane helices 3 (TM3) and 6 (TM6) (2). These helical movements in turn alter the conformation of the intracellular loops of the receptor to promote activation of associated heterotrimeric G proteins.Mutagenesis studies (3-5) demonstrated that the third intracellular loop (i3) mediates a large part of the coupling between receptor and G protein. i3 loops expressed as minigenes have also been shown to directly compete with ␣1B-adrenergic receptors for G q binding (6). Okamoto and colleagues (7) localized a G protein activator region in the C-terminal end of the third cytoplasmic loop of the human ␤2-adrenergic receptor. They showed that a soluble peptide corresponding to this region (R 259 -K 273 ) activates G s protein under cell-free conditions. Moreover, related peptides found in wasp venom, such as mastoparan, stimulate GDP-GTP exchange from purified G proteins (8). These amphiphilic cationic peptides act in the absence of receptors to directly stimulate G i and G o and compete with intact receptor for the G protein ␣ subunit (9). However, there are currently no effective strategies to directly study the mechanism of receptor-G protein coupling in a controlled fashion under in vivo conditions.Here, we present an approach to study receptor-mediated G protein activation by using palmitoylated peptides as receptormodulating agents based on the i3 loops of the protease-activated receptors (PAR), PAR...

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. We previously identified a rho enhancer (RhoBAD) that is active within a subset of abdominal SOP cells to induce larval oenocytes and showed that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor. Here, we show that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, we propose a model for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression.

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. Here, we show that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, we show that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and do so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, we demonstrate that Ato stimulates abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, we also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen.