Abstract
The mechanisms involved in transcriptional regulation by growth hormone (GH) remain unknown. We report here that GH receptor immunoreactivity can be demonstrated in the nuclei of GH-responsive rat and rabbit tissues at both the light and electron micrograph level using monoclonal antibodies to the receptor extracellular domain. Nuclear staining is heterogeneous and associated with both chromatin and the nuclear membrane. To confirm these observations, nuclei were isolated from rabbit liver by two methods, one involving extensive nonionic detergent washes. Scatchard analysis of nuclear fractions revealed high affinity somatogenic receptor in nuclear membranes, nucleoplasm, and chromatin fractions. A panel of GH receptor monoclonal antibodies was used to further define these nuclear binding sites as being antigenically identical to microsomal receptor in all but one case. In addition, affinity cross-linking experiments showed the somatogenic binding subunit to have a reduced Mr of 67,000, similar to the Mr of the GH binding protein. We propose that the association of a GH binding protein with the nucleus may provide a means whereby GH can regulate the transcription of specific genes either directly or indirectly through nuclear kinase C activation. This speculation is congruent with the recent demonstration of a GH response element by Yoon et al. (Yoon, J. B., Berry, S. A., Seelig, S., and Towle, H. C. (1990) J. Biol. Chem. 265, 19947-19954).
Highlights
The mechanisms involved in transcriptional regula- unknown
It was hoped that elucidation of the GH receptor tion by growth hormone (GH) remain unknown
Four distinctnuclear staining patterns were observed on light microscopy: A, no nuclear immunoreaction; R, nuclear immunoreaction associated with the nuclear envelope; C, nuclearimmunoreaction associatedwith both the nuclearenvelope and chromatin(mostly heterochromatin); and D, dense nuclear immunoreaction with no euchromatin/heterochromatin distinction
Summary
HjO, in phosphate-buffered saline (PBS) for 15 min a t 20 "C; (b) elimination of nonspecific protein binding by incubation at 20 "C with 10% normal goat serum for 1 h; (c) incubation overnight a4t "C with mouse anti-GH receptor monoclonal IgG (100 pg/ml (mAbs 1, 5, 7,43, and Brucella) or 25 pg/ml (mAb 263) in PBS, 1%bovine serum albumin (BSA)); (d) incubation with goat anti-mouse biotinylated IgG (diluted 1:150 in PBS, 1%BSA) for 2 h a t 25 "C, ( e ) incubation with avidin (streptavidin)-biotin horseradish peroxidase suspended either in ml of homogenization buffer minus 0.1% Triton X-100 or in100volumes of homogenization buffer with 2% v/v Triton X-100 for preparation of chromatin.
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