FP01-1 Development of a Luciferase Complementation Assay to Investigate GH Receptor Dimerization

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP01-Cell Specific GH & IGF-1 Signaling
Saturday, June 15, 2013: 11:00 AM-11:30 AM
Presentation Start Time: 11:00 AM
Room 134 (Moscone Center)

Poster Board SAT-88
Ying Liu*1, Philip Alton Berry2, Yue Zhang2, Jing Jiang2, Kurt R. Zinn1 and Stuart Jonathan Frank1
1Univ of Alabama at Birmingham, Birmingham, AL, 2University of Alabama at Birmingham, Birmingham, AL
Growth hormone receptor (GHR) and prolactin receptor (PRLR) are single membrane-spanning proteins in the cytokine receptor superfamily. In humans, GH binds and activates both GHR and PRLR, while PRL binds and activates only PRLR. Structural and biochemical studies, as well as FRET and BRET data, suggest GHR and PRLR each form homodimers that may be modified by ligand binding. We previously demonstrated GH-dependent disulfide linkage of GHR homodimers (1), as well as association between GHR and PRLR that is enhanced by GH (2) in human cells that endogenously express these receptors. With the goal of better understanding the mechanism(s) and significance of GHR-PRLR association, we are developing a split luciferase complementation assay. Herein, we report our initial results. For studies of protein-protein association, the luciferase complementation assay tests the ability of expression of one protein molecularly fused to an N-terminal fragment of luciferase to be close enough to the second protein fused to a C-terminal fragment of luciferase to allow reconstitution of intact luciferase activity and measurement of bioluminescence as a report of the closeness of the two proteins. The assay features great sensitivity and specificity with a very low background signal. GHR-GHR homodimerization was initially examined with this system. We created chimeras that included either full-length human GHR (GHR) or human GHR truncated after residue 322 in the proximal cytoplasmic domain (TGHR). Each was fused with either Nluc (residues 1-398 of firefly luciferase) or Cluc (luciferase residues 394-550) with a flexible peptide linker between the GHR and luc portions. Plasmids encoding each fusion or a control with GHR fused to full-length luciferase were transfected alone or in combination into the JAK2-expressing, GHR-deficient human fibrosarcoma cell line, gamma2A-JAK2. Expression of each protein was verified by immunoblotting and the response to GH with JAK2 and/or STAT5 phosphorylation validated that each chimera achieved cell surface presentation. To date, we have confirmed specific luciferase complementation with coexpression of GHR-Nluc/GHR-Cluc and TGHR-Nluc/TGHR-Cluc. As a negative control, coexpression of GHR-Nluc with an estrogen receptor-Cluc fusion yielded no signal. Stronger complementation was observed with TGHR-Nluc/TGHR-Cluc than with GHR-Nluc/GHR-Cluc coexpression, in agreement with published FRET/BRET data. The effect of GH on these complementation signals is currently being evaluated and analogous PRLR fusions are being constructed. These studies should allow fine mapping of GHR-PRLR association determinants.

(1) Frank, S.J., et al., Endocrinol 1994; 135:148. (2) Xu, J., et al., Mol Endocrinol 2011; 25:597.

Nothing to Disclose: YL, PAB, YZ, JJ, KRZ, SJF

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH R01 DK58259 (to SJF)
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