Category: Assay Development and Screening
G-protein Couple Receptors (GPCRs) represent the largest superfamily of membrane proteins and are one of the most pharmaceutically proven drug targets. Calcitonin receptors belong to an important family of GPCRs. The expression and specific pairing of Calctonin (CT) and Calcitonin Receptor-like (CALCRL) receptor subunits with each of the 3 receptor-activity-modifying proteins (RAMP1, 2, 3) play an important role in the pathophysiology of the heterodimers in a variety of disease pathways including Alzheimer’s, pain, heart failure and Type 2 Diabetes. Developing specific in vitro assays for this complex receptor family is paramount for identifying new chemical entities relevant to the treatment of intended diseases. In this report, we will share our findings in our development of specific cell lines for high-throughput screening assays for Amylin (AMY1, 2, 3), Calcitonin Gene-Related Peptide (CGRP), and Adrenomedullin (AM1, 2) receptors. HEK293T and CHO-K1 parental cells were first co-transfected with CT or CALCRL plus RAMP1, 2 and 3 and tested in Gas cAMP functional assay using endogenous ligands. In contrast to previously published reports by other laboratories, we learned that HEK293T cells exhibit high level of endogenous activities when either CT or CALCRL receptor was expressed alone. This background activity was absent in CHO-K1 cells when either of the GPCR subunit was expressed at low level, which laid the basis for our Calcitonin family stable cell line development. We also observed that increased levels of receptor surface expression of the CT subunit alone can render endogenous cAMP activity induced by a-CGRP full agonist. The dynamic interactions between CT/CALCRL and RAMPs presented challenges in developing specific cellular assays, which we overcame by focusing on receptor expression and succeeded in developing well-characterized stable cell lines for each dimeric receptor in the Calcitonin family.
Lisa Minor– Business Development Consultant, Multispan, Inc., Hayward, CA
Business Development Consultant
Lisa K. Minor, Ph.D.
Dr. Minor has been an Executive Consultant at Multispan Inc. since 2011. She is a well recognized scientific expert with 25 years of experience in drug discovery encompassing several therapeutic area research, high throughput screening and safety profiling through her tenure at Johnson and Johnson Pharmaceuticals. She is the editor for Assay Guidance Manual for NIH, served for 8 years as a member of the advisory board for the Society for Biomolecular Sciences (now Society for Laboratory Automation and Screening) and Scientific Advisory Board for the National Toxicology Program, chaired multiple international assay development meetings. She has been consulting for Multispan since January, 2012. Dr. Minor received her postdoctoral training at the Medical College of Pennsylvania and obtained her Ph.D. in Biological Chemistry from the Pennsylvania State University.