Investigating luteinizing hormone receptor signal transduction using the cyclic AMP reporter ICUE1

Mechanisms involved in initiation of signaling by LH receptors have been under active investigation because they play important roles in human fertility and development of gonadal tumors. Assay of cAMP levels in response to hormone treatment is usually used to demonstrate LH receptor activation and has historically relied on biochemical methods. ICUEl is an Epac-based cAMP reporter which undergoes conformational changes after binding cAMP. Unlike traditional biochemical assays, ICUEl combined with FRET techniques is capable of real-time monitoring of cAMP levels in individual cells. In this project, Epac reporters have been used to evaluate LH receptor activity in cells expressing constitutively-active LH receptors or cells where transactivation of LH receptors is reported to occur. For the investigation of constitutively active LH receptors, DNA of ICUEl and yoked LH receptor were co-transfected into CHO cells and expressed on the cell membrane. For the study of LH receptor trans-activation, CHO cells were developed using two plasmids encoding LH receptors with defects either in ligand binding or coupling of LH receptor to Gs. Our results show ICUEl offers an useful tool for evaluating cAMP levels in real-time using single cell imaging methods. Further application of this technique to studies evaluating cAMP level in cells where ligand binding to receptor can be visualized using, for example, quantum dots or nano-gold particles linked to individual molecules of hCG, will be of interest as will studies of cAMP levels during LH receptor desensitization and resensitization in response to brief pulses of hCG.

Name thesis:degree_name

Master of Science (M.S.)

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Cell and Molecular Biology

Grantor dc:publisher

Colorado State University. Libraries

Year dc:date.issued

2010

Authors dc:creator

• Huang, Xin, author
• Roess, Deborah A., advisor
• Barisas, B. George, committee member
• Crans, Debbie C., committee member

• Luteinizing hormone -- Receptors
• Cyclic adenylic acid

Statement dc:rights

• Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.

Language dc:language.iso

eng, English

Identifier URI

https://doi.org/10.25675/3.018605

OAI identifier oai:identifier

oai:mountainscholar.org:10217/234747