Subdomain 2 (S2), Not the Transmembrane Domain (TMD), Determines the Dimerization Partner of Growth Hormone Receptor (GHR) and Prolactin Receptor (PRLR)

Presentation Number: SAT 428
Date of Presentation: April 1st, 2017

Ying Liu*1, Bradford Lepik1, Jing Jiang1, Kurt R. Zinn2 and Stuart J Frank3
1University of Alabama at Birmingham, Birmingham, AL, 2Univ of Alabama at Birmingham, Birmingham, AL, 3University of Alabama at Birmingham/Veterans Affairs Medical Center at Birmingham, Birmingham, AL


Growth hormone (GH) and prolactin (PRL) are peptide hormones secreted mainly by the anterior pituitary gland and sometimes locally. GH receptor (GHR) and PRL receptor (PRLR) are homologous transmembrane proteins in the class I cytokine receptor family. In humans, GH can interact with GHR homodimers or PRLR homodimers and PRL interacts with only PRLR homodimers; multiple signaling pathways are thus triggered, including JAK2-STATs, PI3 kinase, and ERKs. Both GH and PRL signaling have been implicated in aspects of tumorigenicity in animals and humans, particularly in autocrine/paracrine modes [1,2]. In human T47D breast cancer cells endogenously expressing both GHR and PRLR, GHR and PRLR physically associate independent of ligand binding, as determined by specific coimmunoprecipitation [3]. Using split luciferase complementation, we hypothesized that the GHR/PRLR assemblage is arranged as a hetero-multimer comprised of GHR-GHR homodimers and PRLR-PRLR homodimers ((GHR-GHR)x/(PRLR-PRLR)y, where x and y reflect the relative abundance of GHR and PRLR , respectively) [4,5]. Understanding mechanisms that regulate GHR/PRLR assemblages and their potentially diverse signaling consequences is of translational importance. In the current study, we dissect receptor structural determinants for GHR/PRLR homodimerization vs hetero-association. Both GHR and PRLR have extracellular domains (ECDs) composed of the N-terminal subdomain 1 (S1), to which ligand binds, and the more membrane-proximal subdomain 2 (S2), which fosters receptor-receptor contact. Based on previous reports about the roles of S2 vs. the transmembrane domain (TMD) in GHR dimerization, we made GHR(PRLRS2), GHR(PRLRS2-TMD) and GHR(PRLRTMD) chimeras, replacing GHR’s S2 alone, S2 plus TMD, and TMD alone with PRLR’s counterpart respectively. We tested the ability of these chimeras to homodimerize or hetero-associate with WT GHR, using the split luciferase complementation assay. The rationale behind this technique is to split firefly luciferase into two fragments (Nluc and Cluc), each without any enzyme activity alone, and tether them to the tails of two receptors. The two fragments restore luciferase enzyme activity when being brought close to each other by the receptors. The real time profile of ligand-induced complementation change potentially reflects the dimer or multimer arrangement of receptors. By side-by-side comparison among the combinations between GHR-Nluc or GHR(PRLRS2)-Nluc and GHR-Cluc, PRLR-Cluc, GHR(PRLRS2)-Cluc, GHR(PRLRS2-TMD)-Cluc and GHR(PRLRTMD)-Cluc, we found GHR(PRLRS2) and GHR(PRLRS2-TMD) behaved as if PRLR, while GHR(PRLRTMD) behaved as if GHR with regard to their dimerization partners. Thus, we conclude that S2 of GHR and PRLR, rather than their TMDs, determines their dimerization partner. Functional implications of these findings will be discussed.


Nothing to Disclose: YL, BL, JJ, KRZ, SJF