title: Membrane reinsertion of a myristoyl-peptidyl anchored extracellular domain growth hormone receptor.
abstract: The actions of GH are mediated through a cell surface cytokine receptor. We previously demonstrated that naturally occurring truncated membrane bound GH receptors (GHRs) can block GH receptor signaling. We have now investigated whether recombinant extracellular GHR can be conjugated to a myristoylated-peptide (mp) tail and inserted into cell membranes to modulate GHR signaling. Recombinant human extracellular domain (1-241) GHR was expressed in Escherichia coli, purified, and refolded from cell lysate. The free C-terminal cysteine was then reduced and conjugated to an activated preformed mp tail. The properties of the purified tailed GHR (GHR-mp) were then compared with those of the untailed purified GHR 1-241. Fluorescence-activated cell sorter analysis and cell surface binding assays demonstrated that GHR-mp inserted into the cell surface membranes of CHO cells, whereas untailed GHR 1-241 showed no insertion. In a cell-based bioassay GHR-mp partially inhibited wild-type GHR signaling, whereas GHR 1-241 had no effect. Truncated extracellular domain GHR can, when specifically modified with a membrane-localizing mp unit, insert into cell surface membranes and modulate GHR signaling.
authors.affiliation: University of Sheffield, Room 112 Floor M, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom.
authors.forename: C E
authors.initials: CE
authors.lastname: Bowles
authors.forename: I
authors.initials: I
authors.lastname: Wilkinson
authors.forename: R A G
authors.initials: RA
authors.lastname: Smith
authors.forename: A J G
authors.initials: AJ
authors.lastname: Moir
authors.forename: H
authors.initials: H
authors.lastname: Montgomery
authors.forename: R J M
authors.initials: RJ
authors.lastname: Ross
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine; D004251:DNA Transposable Elements; D005110:Extracellular Space / Q000378:metabolism; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010720:Phosphines / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D003545:Cysteine; D004251:DNA Transposable Elements; D013006:Growth Hormone; D010446:Peptide Fragments; D010720:Phosphines; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; C517401:growth hormone receptor (1-241); C080938:tris(2-carboxyethyl)phosphine