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Figure 1 rhNRG-1 construction, synthesis, and erbB-activating activity. (A) Schematic of full-length membrane-bound form of neuregulin (NRG)-1 showing the immunoglobulin-like (IgG-L), and epidermal growth factor-like (EGF-L) regions of its extracellular domain as well as its transmembrane domain (TM) and cytoplasmic tail (CT). The construct used here (designated receptor-active 61-residue recombinant neuregulin-1 peptide [rhNRG-1]), encoding the EGF-like region of NRG-1, encompassing residues Ser177 to Glu237, followed by a stop codon (indicated) and a stroke volume 40 poly A+ tail (open box), was expressed in E. coli from a T7 promoter (arrow). (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (15%) fractionation of E. coli-expressed rhNRG-1 showing the resulting 6.7 kDa Coomassie-stained rhNRG-1 protein at a purity >95% (left lane) and molecular weight standards (right lane). (C) Purified rhNRG-1 stimulated erbB2 and erbB4 phosphorylation in cultured neonatal cardiomyocytes. Cells stimulated with vehicle or with full-length NRG-1 extracellular domain (ECD) (12) were used as negative and positive control subjects, respectively. (D) In vivo activity of rhNRG-1 was similarly evaluated by immunoprecipitation (IP, anti-erbB4 antisera) and immunoblotting (IB, anti–phospho-tyrosine, p-Try, antiserum [Sigma Biochemical, St. Louis, Missouri] or anti-erbB4 antiserum) of lysates prepared from the hearts of mice given 10 µg/kg rhNRG-1, IV, 10 or 20 min before being killed (upper panel). Holo-erbB4 visualized by anti-erbB4 IB is also shown (lower panel). (E) In vivo activation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases was evaluated by IP and IB of lysates prepared from the hearts of mice given rhNRG-1, 10 µg/kg IV, 0, 10, 30, 90, and 270 min before being killed, with anti–phospho-ERK- (p-ERK) (upper panel; 42 and 44 kD isoforms are shown) and anti–holo-ERK- (lower panel) specific antibodies (Santa Cruz Biotechnology, Santa Cruz, California). (F) The p-ERK bands in (E) were quantitated by PhosphorImager analysis and expressed as the fold-increase (stimulation) over the basal (0 min) levels. The time-dependent increase in p-ERK44 is shown. Increased pERK1/2 mitogen-activated protein kinases (upper panels; lower panels show holo-ERK) were also observed in the lysates of the hearts of rats (G) and dogs (H) 10 min after bolus IV administration of rhNRG-1, 10 and 3 µg/kg, respectively.
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