Proteomics analysis of growth hormone isoforms in the human pituitary

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.     

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.     

The functional importance of multiple actin isoforms

Actin is a protein that plays an important role in cell structure, cell motility, and the generation of contractile force in both muscle and nonmuscle cells. In many organisms, multiple forms of actin, or isoactins, are found. These are products of different genes and have different, although very similar, amino acid sequences. Furthermore, these isoactins are expressed in a tissue specific fashion that is conserved across species, suggesting that their presence is functionally important and their behavior can be distinguished quantitatively from one another in vitro. In muscle cells, they are differentially distributed within the cell and some are specifically associated with structures such as costameres, mitochondria, and neuromuscular junctions. There is also good evidence for specific isoactin function in microvascular pericytes and in the intestinal brush border. However, the necessity of specific isoactins for various functions has not yet been conclusively demonstrated.     

The influence of age on human pancreatic growth hormone releasing hormone stimulated growth hormone secretion

63 non-obese healthy subjects aged 18 to 95 years were investigated for age-dependence of GHRH-stimulated GH-secretion. In addition, priming of GH-secretion with three oral doses of propranolol (3 x 80 mg, the last dose 2 hours prior to the second GHRH-bolus) was carried out in 15 subjects below 40 years and 13 subjects older than 70 years. We found that mean maximal incremental GH-levels were inversely correlated with chronological age (r = -0.44, P = 0.001) of the probands. Propranolol premedication caused a significant rise of both basal and peak GHRH-induced relative increases in all subjects tested, whereas GHRH-induced relative increases of GH remained unchanged. In a well selected group of non-obese healthy subjects stimulated GH-secretion is found to undergo an aging process that is supposed to be of pituitary and suprapituitary origin. Priming GH-secretion with a beta-Blocker is possible both in young and very old healthy subjects and is likely to affect the basal GH secretory tone and not GHRH-stimulated GH-secretion.     

Biological activities of two porcine growth hormone-releasing hormone receptor isoforms

Binding of growth hormone-releasing hormone (GHRH) to two isoforms (G3R and G5R) of the porcine GHRH receptor was studied. Both G3R- and G5R-cDNA were isolated from a porcine anterior pituitary cDNA library and have an identical primary structure from aa 1 to 418 and a different aa sequence from aa 419 to 423. In addition, the G5R isoform contains an extra C-terminal tail of 28 aa. The G3R and G5R mRNAs arise from alternative splicing of a single precursor mRNA for GHRH receptors. A mammalian cell expression vector containing either G3R or G5R cDNA under the regulation of a strong human cytomegalovirus promoter was constructed and used to transfect a human embryonic kidney 293 cell line. Two stable transfectants (293/G3R-4 and 293/G5R-12) were isolated on the basis of high expression of the receptor mRNAs. Both G3R and G5R mRNAs were expressed at similarly high levels in 293/G3R-4 and 293/G5R-12 cells; however, GHRH binding to 293/G3R-4 cells was much greater than that observed for 293/G5R-12 cells. Basal as well as GHRH-stimulated GTPase activity and intracellular cAMP concentration are also significantly greater in 293/G3R-4 cells as compared to 293/G5R-12 cells. We conclude that the modification of GHRH receptor at the C-terminal region hindered GHRH binding to the receptor and thus attenuates its biological activities.     

Tumor- and pregnancy-derived isoforms of human chorionic gonadotropin: biological and diagnostic relevance

OBJECTIVES: Human chorionic gonadotropin (hCG) and hCG variants are of high clinical importance for the diagnosis of pregnancy, monitoring of abnormal and ectopic pregnancies, testing for Down's syndrome or monitoring therapy of hCG-secreting malignancies. In serum and urine, hCG appears in microheterogeneous isoforms with respect to protein backbone structure and the extent of glycosylation. The present study reports on the identification, immunological characterization, biological activity of glycosylation isoforms of pregnancy (preg) and tumor-derived (tu) hCG, and the impact of glycosylation on diagnostic immunoassays. METHODS: Twenty-two urinary preg- and tu-hCG isoforms were separated by preparative isoelectrofocusing (hCG-pI variants) and characterized by Western blot. Number, topography and accessibility pattern of epitopes on their surface was evaluated by two-site radioimmunoassays using 14 different monoclonal antibodies (mabs). Binding of hCG isoforms to four different LH/CG receptors was investigated in radioreceptor assays, and their biological activity determined by measuring cAMP elevation. RESULTS: All 22 hCG glycosylation variants appeared immunologically intact: each isoform, even when highly acidic, expressed all 14 surface epitopes which were arranged in a topographical manner indistinguishable from crude hCG. hCG isoforms were able to bind to four different receptor variants, with slightly varying affinities, but orientations indistinguishable from each other as shown by identical epitope accessibility patterns. Each of the hCG-pI variants was able to activate the LH/CG-Rs, but with varying reactivities. CONCLUSIONS: We conclude that in contrast to deglycosylated hCG, all hCG glycosylation isoforms investigated act as receptor agonists. Moreover, there is no overspecificity of mabs to certain hCG isoforms due to carbohydrate variability that exclude others from diagnostic measurement.     

Divalent metal cation chelators enhance chromatographic separation of structurally similar macromolecules: separation of human growth hormone isoforms

Human GH isoforms were separated by anion-exchange chromatography using a linear NaCl gradient in the presence and absence of EDTA and EGTA. SDS–PAGE showed that glycosylated 24-kDa hGH did not appreciably separate from other hGH variants in the absence of metal chelators. However, in the presence of metal chelators, glycosylated 24-kDa hGH separated from the bulk of the hGH isoforms. Human GH isoforms were also separated by size-exclusion chromatography in the presence and absence of metal chelators. Glycosylated 24-kDa hGH eluted with the bulk of the hGH isoforms in both separations. The inclusion of metal chelators in chromatographic buffers to alter the charge and/or size of proteins by stripping their metals may be a generally useful strategy in their fractionation.     

人生长激素研究进展

综述了人生长激素的结构,及最近几年国内外对人生长激素在分子结构、基因改造、制备、提纯及检测等方面的最新进展和研究趋势,对各种可能影响生长激素分泌的因素进行了分析、讨论。