CHARACTERIZATION OF THE SHORT ISOFORM OF THE GROWTH-HORMONE RECEPTOR SYNTHESIZED BY RAT ADIPOCYTES

Two mRNA transcripts that are believed to be alternately spliced products of the GH receptor gene have been reported in a variety of rat tissues. The smaller (1.2 kilobases) transcript was cloned from an adipocyte library, sequenced, and found to encode a protein identical to the soluble GH-binding protein (GHBP) in plasma. An assay that is specific for the short isoform of the GH receptor, often referred to as the GHBP, has been developed using a rabbit antiserum that recognizes the unique amino acid sequence at its carboxyl end. The assay depends upon immunoprecipitation of a complex consisting of [I-125]human GH, the binding protein, antiserum, and protein-A cross-linked to agarose beads. To validate the assay, samples of rat plasma were analyzed and found to contain sufficient binding protein to bind 1.46 pmol (32 ng) GH/ml, with an affinity of 2.7 X 10(9) M-1. In adipocyte extracts, binding protein activity was sufficient to bind 61 fmol GH/g tissue, with an affinity of 2.3 X 10(9) M-1. The binding protein was found primarily in the particulate fraction of adipocytes, and it is estimated that adipocytes contain approximately 7000 copies of the binding protein/cell. Only 10% of the binding activity was present in the high speed supernatant of adipocyte homogenates, and soluble binding protein did not appear to be released into the incubation medium when adipocytes were incubated in vitro. A 50-kilodalton (kDa) S-35-labeled protein that may be a glycosylated form of the binding protein was immunoprecipitated from both the soluble and particulate fractions of adipocyte extracts by the antiserum, and addition of the synthetic peptide antigen blocked immunoprecipitation of this protein. A 150-kDa protein in the high speed supernatant fraction was also specifically immunoprecipitated by the antiserum. Although it is unlikely to be a glycosylated form of the binding protein, it may cross-react with the antiserum or perhaps be coprecipitated, because it interacts with the binding protein. In addition, 38- and 42-kDa bands were specifically immunoprecipitated from the detergent-treated particulate fraction of adipocyte extracts that were enriched for the binding protein by adsorption to immobilized GH. We conclude that 1) adipocytes synthesize the short isoform of the GH receptor, and that this protein is primarily associated with a membrane fraction of the cells; and 2) the GHBP expressed in adipocytes is not released into the incubation medium and differs in size from the GHBPs in rat plasma. Perhaps, it associates with integral membrane proteins and provides for ligand binding by a more complex GH receptor, which includes one or more additional polypeptides for signal transduction.