Adrian C. Herington

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.

Developmental expression of the growth hormone receptor gene in rabbit tissues

Total RNA from several adult (6-18-month-old) rabbit tissues was characterized using an oligonucleotide probe derived from the extracellular domain of the nucleotide sequence of the rabbit growth hormone receptor (GH-R) cDNA. Multiple GH-R mRNA species of approximately 4.6, approximately 3.3, 2.1 and approximately 1.4 kb were detected. The major 4.6 kb transcript was detectable in all tissues examined but with quite marked abundance differences. The highest level of expression was observed in liver followed closely by muscle. A qualitative assessment of insulin-like growth factor I (IGF-I) mRNA abundance was made in these same tissues. The data showed that the tissue abundance of GH-R mRNA was not necessarily parallel to that of IGF-I mRNA. The ontogeny of GH-R mRNA was studied in rabbit liver, muscle, heart and kidney. Low levels of GH-R mRNA were detectable in all fetal tissues studied except kidney which showed relatively high levels, suggesting that GH may play an important role in early kidney development. The overall developmental pattern of GH-R mRNA was similar in heart, muscle and liver, being low in fetal and early neonatal (day 3) periods and reaching maximal levels between 2 and 6 months. However, in kidney the pattern contrasted markedly. Relatively high levels of GH-R mRNA were observed in fetal and early neonatal (day 3) kidney with little change throughout development. The developmental pattern of IGF-I gene expression was not necessarily co-ordinately regulated with the ontogenic pattern of GH-R gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of messenger RNA for the rat growth hormone receptor and serum binding protein

Little is known of the regulation of gene expression for the family of growth hormone (GH) and prolactin (PRL) receptors (PRL-R). Furthermore, the relationship between expression of the GH receptor (GHR) and its soluble truncated form (GH-binding protein, GHBP) is unclear. The actions of both GH and PRL are developmentally regulated and several studies have examined the ontogeny of these receptors by classical hormone-binding techniques. In the current study we have examined the expression of GHR/GHBP and PRL-R mRNA in the male rat over a broad developmental range--fetal through to 110 days of age. The GHR mRNA (4.5 kb) was barely detectable in fetal and early (less than 20 days) postnatal livers, but was followed by a gradual increase up to 40 days of age by which time adult plateau levels were reached. In contrast, hepatic GHBP mRNA (1.2 kb) was clearly identifiable in the fetus and subsequently followed a similar pattern to the 4.5 kb GHR mRNA although there was a somewhat earlier rise. Hepatic membrane binding studies using 125I-bovine GH as ligand revealed no measurable binding activity at less than 20 days of age. Binding remained low thereafter. In contrast, the serum GHBP binding activity was detectable at 10 days of age and rose to adult levels by 50 days of age. These results indicate that mRNA species for GHR, GHBP, PRL-R and insulin-like growth factor I (IGF-I) are all developmentally regulated with the pattern for IGF-I correlating more closely with that of GHBP than GHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Divergent ileal IGF-I and IGFBP-3 gene expression after small bowel resection: a novel mechanism to amplify IGF action?

Changes in the levels of ileal insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA in the rat following massive small bowel resection (MSBR) have been investigated with a sensitive S1 nuclease assay. IGF-I mRNA levels vary little over 7 days; in contrast IGFBP-3 mRNA levels decreased to one-third 7 h post-MSBR, and remained suppressed for the length of this study. We postulate that decreased ileal synthesis of IGFBP-3 enhances the ability of IGF-I to stimulate the adaptive response.

Identification and characterization of growth hormone receptors on isolated rat adipocytes.

It is now recognized that growth hormones (GH) can exert direct metabolic effects on adipose tissue from hypophysectomized or normal rats in vitro. This implies the presence of receptors for GH in this tissue. The interaction of 125I-labelled human and bovine GH has been studied therefore in isolated adipocytes from male and female rats. Binding of 125I-hGH was dependent on time (with equilibrium being reached by 2 hrs at 23 degrees C) and number of fat cells (binding being linear up to 1.5 x 10(6) cells/tube). Binding was partially reversible (half-time for dissociation being 4 hrs at 23 degrees C) and specific for GH being displaced by various growth hormones but not by lactogens or other hormones. Scatchard analysis revealed linear plots with an affinity (KA) of 0.8 x 10(9) M-1 and 24,000 sites per cell for adipocytes from 36-day old male rats. 125I-labeled bovine GH binding was somewhat lower, being reflected in a slightly reduced binding capacity. No obvious sex difference in hGH binding was evident but there was a quite striking age-dependence of binding, being low prior to weaning, reaching a peak between 24 - 36 days and then declining to low levels again in the adult. The demonstration of specific GH receptors in adipose tissue confirms the notion of direct actions of GH in this tissue and should now permit further studies of the relationship between GH binding and GH action.

Identification of a novel growth hormone binding protein mRNA in rat liver

The presence of highly specific serum binding proteins for growth hormone (GH) has been well characterized in many species. In the rat the major growth hormone-binding protein (GH-BP) is a truncated, variant form of the target tissue GH receptor and is derived by an alternative mRNA splicing event. The GHBP mRNA is coexpressed in all tissues expressing the full length GH receptor. In the present study, we have made an oligonucleotide probe to the unique hydrophilic tail of the rat GHBP mRNA (1.2kb) and identified a novel GHBP-like mRNA of 2.6 kb transcript in addition to the 1.2 kb transcript. This unique 2.6 kb transcript was expressed/detected only in rat liver. There was no significant difference in abundance between the sexes or during pregnancy, implying that this transcript may be regulated independently of the 1.2 kb mRNA. The 2.6 kb transcript was clearly identifiable in the fetus, as was the 1.2 kb transcript, but showed virtually no change in abundance with age, in sharp contrast to the 1.2 kb mRNA, which has a distinct developmental pattern, being low in the fetus and peaking early postnatally. RNAse H treatment suggested that this 2.6 kb transcript is polyadenylated. A corresponding 2.6 kb mRNA has been detected using a longer cDNA or cRNA probe for the GH-binding domain of the rat GHR/GHBP. These data collectively suggest that the 2.6 kb mRNA transcript is a bonafide but tissue-specific GHBP mRNA and that the 1.2 and 2.6 kb mRNAs are likely to differ primarily with respect to the length of the 3 untranslated region of the sequence.

Water Soluble Receptors for Human Growth Hormone from Rabbit Liver

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes form pregnant female rabbits in 1 mM Tris buffer (ph 7.5 or 9.0) at 4 degrees C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (Ka 2.2 x 10(9) M-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (Ka integral of 1 x 10(8) M-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.

Evidence for a biologically active, carrier-bound form of non-suppressible insulin-like activity (nsila) in human serum

1. Quantitative gel filtration of Cohn fraction IV-1 has shown that high MW NSILA recovered under neutral conditions (pH 5.5) was greater in magnitude (approximately X 2) than acid-stable high MW NSILA obtained under acid conditions. 2. Recombination, at neutral poH, of high and low MW NSILA, obtained by previous acid gel filtration, gave quantitative recovery of activity which was solely of high MW. 3. These data indicate that a biologically active, and dissociable high MW form of NSILA exists in serum. 4. Con A-Sepharose affinity chromatography gave one unbound NSILA fraction and two bound fractions, which both gave high and low MW activity after acid chromatography. 5. This study provides the first evidence for a carrier-bound form of low MW NSILA which retains biological activity.

Interaction between the hepatic growth hormone receptor and concanavalin A

The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum

Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000-400,000 with a peak at about MW 200,000. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000-600,000 (77% of the fraction NSILA or 28% of total serum NSILA). Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000-200,000 and 35,000-100,000 fractions and corresponded to 37% of total serum NSILA. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000-100,000. Low MW NSILA (less than 15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000-600,000. This complex was not bound by Con-A Sepharose.