Submitted to: Federation of European Biochemical Societies Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2010
Publication Date: 3/19/2010
Publication URL: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-4YCWNN5-6&_user=2139813&_coverDate=03%2F19%2F2010&_rdoc=24&_fmt=high&_orig=browse&_srch=doc-info(%23toc%234938%232010%23994159993%231783606%23FLA%23display%23Volume)&_cdi=4938&_sort=d&_docanchor=&_ct=35&_acct=C000054276&_version=1&_urlVersion=0&_userid=2139813&md5=83e22ea740145212ac1f3b2c74e12431
Citation: Choi, M.Y., Jurenka, R.A. 2010. Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor. Federation of European Biochemical Societies Letters. 584:1212-1216. Interpretive Summary: Lepidopteran insects are major pests of agriculture throughout the world, and their control is responsible for the largest use of chemical pesticides. Novel biologically-based or environment friendly methods of controlling these pests are needed to reduce our dependence on pesticides. Insect neuropeptides play many critical roles in insect development and metamorphosis. Identifying and understanding the mode of action for these neuropeptides and their receptors could lead to new non-insecticidal control methods. Scientists at the Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, FL, and the Department of Entomology, Iowa State University, Ames, Iowa, have characterized the PBAN (pheromone biosynthesis activating neuropeptide) and receptor activation process through modeling. The PBAN receptor (PBAN-R) is a G-Protein coupled receptor (GPCR) consisting of 7-transmembrane domains that form three (3) extracellular loops on the cellular surface. These loops create binding pocket(s) for the specific ligand, PBAN in the case of PBAN-R. Using chimeric GPCRs we proposed that the third extracellular loop 3 (ECL-3) is a critical binding site needed to activate the receptor. In the present study, we characterize the 3rd extracellular domain of PBAN-R, simulating three site-directed point mutations and PBAN activation of these mutant receptors, and predicted conformational structure. Results are discussed in context of the structural features of PBAN-R that are required for receptor activation as compared to PBAN-R mutants using receptor activation experiments and computational modeling. The research results represent a step toward the goal of developing novel insect control methods by finding agonists and/or antagonists for PBAN/pyrokinin receptors. This is the first study using mutant receptors and predictive modeling in insect GPCRs.
Technical Abstract: Insect neuropeptides are produced in the central or peripheral nerve tissues, and released to regulate various physiological and behavioral actions during development and reproduction. Pheromone biosynthesis-activating neuropeptide (PBAN)/Pyrokinin is a major neuropeptide family characterized with a common FXPRLamide or similar penta-peptide in C-terminal sequence. PBAN is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors for this peptide family were first identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. Like most GPCRs, PBAN receptor (PBAN-R) activation occurs with a proper conformational change after PBAN interacts with specific binding domains. Little information is known about the structure of peptide hormone GPCRs in insects other than primary sequence information. Testing of chimeric receptors, created from distant but related GPCRs, is a useful strategy to understand how specific receptors transduce agonist binding into receptor activation. We have characterized the PBAN-R active binding domains using chimeric GPCRs and proposed that the extracellular loop 3 (ECL-3) is a possible binding site for determining ligand selection and activation of the receptor. In the present study, we continued to characterize the 3rd extracellular domain of PBAN-R through three site-directed point mutations and PBAN activation of these mutant receptors, and predicted conformational structure. Results are discussed in context of the structural features of PBAN-R that are required for receptor activation as compared to PBAN-R mutants using receptor activation experiments and in silico computational modeling. This first case study will help characterize these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors, an important class of receptors in insects.