Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

Recombinant-derived chicken growth hormone used for radioimmunoassay

The use of recombinant-derived chicken growth hormone (rcGH) in an avian growth hormone (GH) radioimmunoassay (RIA) procedure is described. Antiserum to turkey GH bound 125I-labeled rcGH, and unlabeled rcGH or turkey GH displaced binding in a dose-related manner. The dose-response curves of sera and pituitary extract from chickens and turkeys were parallel to the rcGH standard curve. Sera from hypophysectomized (hypox) chickens and turkeys produced no dose-response and did not inhibit binding of labeled rcGH. Recovery of rcGH added to hypox sera was quantitative. Modification of the homologous turkey GH RIA protocol of Proudman and Wentworth (1) to use rcGH made possible either an increase in assay sensitivity or a 3-day reduction in incubation time.     

Phosphorylation of the chicken progesterone receptor

We have examined the phosphorylation of the chicken progesterone receptor in tissue slices and in vitro. The receptor is phosphorylated in tissue slices and this phosphorylation is stimulated by progesterone. As others have reported, partially purified receptor preparations contain a kinase activity which phosphorylates histones and receptor. We have shown that this activity can be separated from the receptor. The receptor is a substrate for several kinases, including the catalytic subunit of the cAMP-dependent protein kinase and PPdPK, a polypeptide-dependent protein kinase. Phosphorylation by the cAMP-dependent protein kinase results in an apparent increase in the molecular weight of the receptor when the receptor is analyzed by SDS-PAGE. These results are consistent with apparent changes in molecular weight observed for rabbit and human progesterone receptor upon treatment of tissue or cells with hormone.     

Novel AluI-polymorphism in the fourth intron of chicken growth hormone gene

A novel AluI-polymorphism in the fourth intron of chicken growth hormone gene was shown. The cytosine to thymine transition in the restriction site for AluI was detected. Primers flanking the 460 bp fragment of the fourth intron containing a polymorphic restriction site for AluI were designed. The nucleotide sequence fragments amplified polymorphic variants were determined. Using designed primers, the genetic structure of populations of White Plymouth Rock, Poltava Clay, Rhode Island Red, and Borkovskaya Barvistaya chicken breeds were analyzed. It was found that growth hormone gene (by AluI-polymorphism in the fourth intron) was polymorphic in all experimental populations. Frequencies of alleles C and T in chicken population of White Plymouth Rock breed were 0.14 and 0.86, Rhode Island Red were 0.3 and 0.7, Poltava Clay were 0.04 and 0.96, and Borkovskaya Barvistaya were 0.08 and 0.92, respectively. The tendency to increase egg production and egg weight of chicken with C/C genotype and meat quality (live weight, carcass weight, weight of pectoral muscles) of chickens with genotype T/T of Rhode Island Red chicken breed was shown.     

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

Prolactin and growth hormone gene polymorphisms in chicken lines of Ukrainian selection

Prolactin and growth hormone gene polymorphisms were studied in two Ukrainian meat-egglaying (G-2) and egg-laying (A) chicken lines. Polymorphism was revealed by the presence of an insertion in the prolactin promoter, by single nucleotide polymorphism (SNP, C to T transition at position ?2402) of the prolactin gene, and in the first intron of the growth hormone gene. The frequency of alleles I and D in chicken line G-2 was 0.135 and 0.865; in chicken line A, 0.730 and 0.270; that of alleles C and T, 0.155, 0.845 and 0.745, 0.255; that of alleles A, B, and C, 0.435, 0.395, 0.170 and 0.670, 0.160, 0.170, respectively. It is shown that an increased frequency of alleles I, C, and A in line A corresponds to egg-laying hens.     

High diversity of the chicken growth hormone gene and effects on growth and carcass traits

The chicken growth hormone (cGH) gene plays a crucial role in controlling growth and metabolism, leading to potential correlations between cGH polymorphisms and economic traits. In this study, DNA from four divergent chicken breeds were screened for single nucleotide polymorphisms (SNPs) in the cGH gene using denaturing high-performance liquid chromatography and sequencing. A total of 46 SNPs were identified, of which 4 were in the 5' untranslated region, 1 in the 3' untranslated region, 5 in exons (two of which are nonsynonymous), with the remaining 36 in introns. The nucleotide diversity in the cGH gene ( theta = 2.7 x 10(-3)) was higher than that reported for other chicken genes, even within the same breeds. The associations of five of these SNPs and their haplotypes with chicken growth and carcass traits were determined using polymerase chain reaction-restriction fragment length polymorphism analysis in a F2 resource population cross of two of the four chicken breeds (White Recessive Rock and Xinghua). This analysis shows that, among other correlations, G+1705A was significantly associated with body weight at all ages measured, shank length at three of four ages measured, and average daily gain within weeks 0 to 4. Thus, this cGH polymorphism, or another polymorphism that is in linkage disequilibrium with G+1705A, appears to correspond to a significant growth-related quantitative trait locus difference between the two breeds used to construct the resource population.     

The effect of immunization against somatostatin on growth rates and growth hormone secretion in the chicken

The effect of both active passive immunization against somatostatin on growth rate and growth hormone levels was studied in chickens. Passive immunization against somatostatin by administration of antiserum had no effect on rate of growth of chickens and no persistent effect on circulating growth hormone (GH) levels. In acute experiments, administration of anti-somatostatin serum caused a marked elevation of GH levels in chickens at both 4 and 8 weeks of age, but the relative stimulation was greater in the older birds. Active immunization against somatostatin significantly stimulated growth rate in chickens, but was not shown to have a clear effect on circulating GH levels. These data suggest that somatostatin control over GH secretion may not be fully developed in the chicken at 4 weeks of age, but that immuno-neutralization of somatostatin can produce an increase rate of growth in chickens similar to that seen in mammals.     

Phosphorylation heterogeneity of tryptic phosphopeptides of chicken riboflavin-binding protein

The tryptic phosphopeptide of hen egg white riboflavin-binding protein has been found to exist as a mixture of peptides which differ only with respect to the number of covalently bound phosphoryl groups. Anion-exchange chromatography was used to separate homologues of the tryptic phosphopeptide of egg white riboflavin-binding protein. Four peptide peaks were obtained and analyzed using plasma desorption mass spectrometry. Molecular ions obtained agree closely with calculated molecular weight values for phosphopeptides with 8, 7 and 5 phosphoryl groups. Amino acid analyses showed that the octa- and hepta-phosphorylated peptides were pure and had the same amino acid compositions.     

Phosphorylation of iodopsin, chicken red-sensitive cone visual pigment

The amino acid sequence has been determined for the carboxyl-terminal 41 amino acids of chicken red-sensitive cone pigment, iodopsin. This sequence is distinct from but structurally homologous to that of other visual pigments. It contains a region rich in the hydroxy amino acids serine and threonine. In the related rod cell visual pigment, rhodopsin, such serines and threonines have previously been identified as sites for phosphorylation by rhodopsin kinase. Phosphorylation of photolyzed rhodopsin serves to terminate its ability to function in visual transduction as an activator of G-protein. We have purified and reconstituted both chicken rhodopsin and chicken iodopsin and shown them to be phosphorylated by bovine rhodopsin kinase. Chicken iodopsin has a Km and Vmax similar to but distinguishably different from that for bovine rhodopsin. These results, in conjunction with other data, suggest that visual pigments in cone cells, upon absorption of light, undergo functional processes similar to those of the visual pigments in rod cells.     

Heterogeneity of chicken growth hormone (cGH). Identification of lipolytic and non-lipolytic variants

The identification and biological activity of chicken growth hormone (cGH) charge variants is described. On the basis of electrophoresis and immunoreactivity chicken pituitary glands contain at least two "charge" variants (Rf = 0.22 and 0.3) which have different net charge but similar molecular weight (26,300 d). Both are immunoreactive but show different bioactivity with adipose explants, band 0.22 being lipolytic whereas band 0.3 appears to be inactive. The abundance of these cGH bands vary with age, both being higher in young birds and lower in adults. These results suggest that cGH variants may have different biological actions.     

Chronic intravenous infusion of chicken growth hormone increases body fat content of young broiler chickens

The purpose of this study was to determine the effects of programmed intravenous infusion of chicken growth hormone (cGH) on growth and metabolism of young broiler chickens (4–7 weeks of age). Four-week-old broiler cockerels, fitted with indwelling jugular catherters, were randomly assigned to three treatment groups (6 birds/group): pulsatile infusion of buffer (phosphate buffer, pH 7.4)[PB-P] at 3 hr intervals, pulsatile infusion of cGH (15 μg/kg at 3 hr intervals)[GH-P], or continuous infusion of cGH (120 μg/kg-day)[GH-C]. Birds were bled 5 min before (0-min) and 5 min post-infusion (relative to the pulses of PB and cGH) at 5, 6, and 7 weeks of age. Pulsatile infusion of cGH increased (P < 0.05) feed consumption by 24% and reduced (P < 0.05) feed efficiency by 14% without affecting body weight (BW) gain. The relative weights (%BW) of liver, abdominal fat, and bursa of Fabricius were not affected by the pattern of cGH infusion. However, the body fat content of cGH-infused chickens was increased (P < 0.05) by 13% (GH-C) and 17% (GH-P), while body protein and water contents were slightly reduced. Body ash content was not affected by pattern of cGH infusion. When compared with the PB-P controls, the GH-P treatment depressed (P < 0.05) hepatic GH-binding activity by 52% without affecting plasma insulin-like growth factor-I (IGF-I) levels. Continuous infusion of cGH increased (P < 0.05) plasma IGF-I by 16%, thyroxine (T₄) by 31%, and glucagon levels by 55%, although plasma GH levels were only 47% higher than those of the PB-P group. However, the GH-P treatment was only half as effective as the GH-C pattern in elevating plasma levels of T₄ and glucagon. This study shows that programmed intravenous infusion of cGH increases deposition of body fat in young rapidly-growing broiler chickens.     

Dosing of growth hormone in growth hormone deficiency

Growth hormone (GH) treatment of GH-deficient (GHD) children is to a certain extent standardized worldwide. Recombinant 22 kDa GH is injected once daily by the subcutaneous route, mostly in the evening. The amount of GH injected (calculated per kg body weight or body surface area, expressed in terms of IU or mg) in prepubertal children mimics the known production rate (approximately 0.02 mg [0. 06 IU]/kg body weight per day). However, there is a wide variation in dosage, the reasons for which are partly unknown and partly due to national traditions and regimes imposed by authorities regulating reimbursement. The situation during puberty is less standardized, with most clinicians still not increasing the dosage according to known production rates. The results of these approaches in terms of adult height outcome are not always satisfactory. In order to achieve optimal height development during childhood, puberty and adulthood, strategies must be developed to individualize GH dosing according to set therapeutical goals taking into account efficacy, safety and cost. The implementation of prediction algorithms will help us to reach these goals. In addition, other response variables will have to be monitored during treatment in order to correct for deficits resulting from GHD.     

Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase.

Phosphorylation of immunopurified chicken oviduct progesterone receptor (PR) was studied in intact cells and under cell-free conditions. Cytosol PR was isolated by incubation with anti-PR monoclonal antibody alpha PR22 adsorbed to protein A-Sepharose and suspended in a reaction mixture containing 10 mM Mg2+, 0.1 mM [gamma-32P]ATP, and the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) from bovine heart. All three major proteins of avian PR (PR-A, 79 kDa; PR-B, 110 kDa; 90 kDa) incorporated 32P-radioactivity on serine residues. The phosphorylation reaction was inhibited by synthetic inhibitors of protein kinases, H-8 and 20-residue peptide IP20. A 40 degrees C preexposure of PR oligomer increased phosphorylation of the 90-kDa protein, known to be a heat-shock protein (hsp-90). The extent of the phosphorylation reaction was temperature-dependent as the 32P-incorporation into PR-A and PR-B increased gradually, showing a maximum at 37 degrees C. Multiple phosphopeptides (4-7) were resolved by two-dimensional electrophoresis chromatography following cleavage of 32P-labeled peptides with trypsin. Both A and B forms of receptor showed similar phosphorylation patterns with B receptor digestion exhibiting two to three additional peptides. Under physiological conditions, preincubation of oviduct mince with forskolin, a regulator of intracellular cAMP levels, caused a greater extent of phosphorylation of PR-A and PR-B proteins. The results of this study demonstrate that chicken oviduct PR is an excellent substrate for the action of cAMP-PK in vitro and that this enzyme may be a physiological regulator of progesterone action in the oviduct.     

Phosphorylation of hormone sensitive phosphodiesterase in isolated adipocytes.

Cyclic AMP phosphodiesterase in rat adipocytes is stimulated by insulin and also by agents that increase cyclic AMP levels. When the enzyme is immunoprecipitated from a solubilised microsomal preparation from adipocytes prelabelled with radioactive phosphate and separated on SDS polyacrylamide gels, label is found in a protein band at the expected Mr for adipose tissue phosphodiesterase. Treatment of the adipocytes with isoproterenol or methyl isobutylxanthine increased the labelling of this band. Insulin alone had no effect on its labelling but did decrease the incorporation of label caused by isoproterenol.     

Isolation and structural characterization of chicken hypothalamic luteinizing hormone releasing hormone

Studies on partially purified chicken hypothalamic luteinizing hormone releasing hormone (LHRH) utilizing chromatography, radioimmunoassay with region-specific antisera, enzymic inactivation, and chemical modification established that the peptide is structurally different from mammalian hypothalamic LHRH. These studies demonstrated that arginine in position 8 is substituted by a neutral amino acid. On the basis of conformational criteria and evolutionary probability of amino acid interchange for arginine, the most likely substitution was glutamine. We therefore synthesized [Gln8]-LHRH and established that it had identical chromatographic, immunologic, and biological properties to the natural chicken peptide. In concurrent studies, purification of 17 micrograms of an LHRH from 249,000 chicken hypothalami was achieved using acetic acid extraction, immuno-affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. Amino acid composition and sequence analyses confirmed the structure of this form of chicken LHRH as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.