Poster abstracts

Poster number 3 submitted by Wei Liu

Functional impact of manipulation in relative orientation of human prolactin receptor domains

Wei Liu (Ohio State Biochemistry Program, The Ohio State University), Charles L. Brooks (Veterinary Biosciences, The Ohio State University)

Abstract:
Hormone binding creates active receptor dimers for class 1 cytokine receptors but the detailed molecular mechanism by which these receptors are activated by their ligands is not well characterized and it is unknown if these receptors share common mechanisms. A rotation model has been proposed for the activation of human erythropoietin receptor (hEPOr) and human growth hormone receptor (hGHr) and is supported by evidence showing that alanine additions at the junction of the transmembrane (TM) and intracellular (IC) domains and/or within the TM domain influenced receptor activities. This evidence suggests that alanine additions changed the relative orientations of the IC domains and their subsequent activation. We wished to determine if a similar mechanism was at play with human prolactin receptor (hPRLr). Up to four alanines were added between the TM and either the IC or extracellular (EC) domains to extend the TM helix and to rotate the IC or EC domains. Also, up to four glycines were placed between the TM and IC domains to provide increased flexibility between these two domains. Wild-type hPRLr or various mutant receptors were expressed in human embryonic kidney 293T cells that express endogenous Janus kinase 2. In the absence of human prolactin (hPRL) none of the alanine or glycine additions increased receptor phosphorylation above that of wild-type hPRLr. In the presence of hPRL both wild-type hPRLr and each of the mutant receptors were successfully phosphorylated. These data do not support a rotation mechanism for hPRLr activation or a requirement of a fixed spatial relationship between the TM and IC domains for hPRLr activation. In a second set of experiments both wild-type hPRLr and either alanine or glycine extended receptors were co-expressed in 293T cells. In the absence of hPRL there was no detectable phosphorylation of hPRLr. Such data do not support a piston movement between the hPRLr pair in their activation.

Keywords: prolactin receptor, human, structure-function