Membrane organization of luteinizing hormone receptors during signal transduction

2010 Summer.Includes bibliographic references (pages 141-157).Covers not scanned.Print version deaccessioned 2022.Mechanisms involved in signal transduction by luteinizing hormone (LH) receptors are important for regulating key events in mammalian reproduction, such as ovulation, sex hormone production and maintenance of pregnancy. Studying the organization of LH receptors in the plasma membrane during hormone-mediated signaling provides insights into the protein interactions needed for important physiological responses. We used biochemical and biophysical methods to examine the role of the plasma membrane in contributing to LH receptor desensitization. Using single particle tracking and sucrose gradient ultracentrifugation, we determined that individual human LH receptors are confined in small membrane compartments and localize in membrane rafts for several hours following desensitization. These receptors do not demonstrate signaling via cyclic adenosine monophosphate (cAMP) while they are confined, suggesting that the microenvironment within these compartments may be different for desensitized versus actively signaling receptors. We also investigated self-association of human LH receptors using homotransfer fluorescence resonance energy transfer (homo-FRET) and fluorescence correlation spectroscopy. We determined that human LH receptors self-associate following desensitization and in response to increasing concentrations of human chorionic gonadotropin (hCG). LH receptors demonstrated the highest degree of aggregation in response to saturating concentrations of 100 nM hCG. Using single particle tracking, we examined whether native LH receptors expressed on KGN human granulosa-like tumor cells, or M l7 human neuroblastoma cells, become confined in small membrane compartments in response to hormone binding. We found that confinement of native LH receptors in small plasma membrane compartments depended on hCG concentration. With increasing concentrations of hCG, more LH receptors became confined in small membrane compartments with an average diameter of less than 100 nm. These receptors also exhibited slower rates of lateral diffusion. We reported the movement of non-functional hormone-receptors, labeled with deglycosylated hCG, into small membrane compartments in response to hCG treatment that saturated other available LH receptors on the membrane. This finding suggests that interactions between functional and non-functional LH receptors may occur in membrane microdomains during signal transduction

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs