scholarly journals Serum and liver cytosolic growth-hormone-binding proteins are antigenically identical with liver membrane ‘receptor’ types 1 and 2

1986 ◽  
Vol 237 (3) ◽  
pp. 885-892 ◽  
Author(s):  
R Barnard ◽  
M J Waters
Keyword(s):  
Growth Hormone ◽  
Binding Site ◽  
Binding Proteins ◽  
Membrane Receptor ◽  
Rabbit Serum ◽  
Receptor Subtypes ◽  
Hormone Binding ◽  
Liver Growth ◽  
Serum Gh ◽  
Gh Receptors

Studies with a panel of monoclonal antibodies (MAbs) reactive towards the presumptive rabbit liver growth-hormone (GH) receptor show that the rabbit serum GH-binding proteins share seven antigenic determinants (three at the hormone-binding site and four located elsewhere) with the liver cytosolic GH-binding proteins and the putative GH ‘receptors’ associated with the hepatocyte membrane. The rabbit serum binding proteins have an affinity for GH similar to the membrane GH receptors [for human GH, Ka = 2.45 (+/- 0.15) X 10(9) M-1 (mean +/- S.E.M., n = 8)] and high capacity relative to membrane ‘GH receptors’. Analogues of the postulated membrane ‘receptor’ subtypes 1 and 2 exist in the serum, but not subtype 3, which is also absent from liver cytosol. The serum and cytosolic binding proteins have identical cation-dependence properties; hGH binding is Ca2+-dependent, whereas oGH binding is Ca2+-independent. Affinity labelling of hGH-affinity-purified serum binding proteins with 125I-hGH demonstrated a major GH-binding subunit, of Mr 55,000, identical with the major component purified from membranes. In view of their high affinity and capacity, the serum binding proteins could control availability of GH to membrane receptors. It is suggested that the cytosolic binding proteins may be newly synthesized serum binding proteins. The existence of a close relationship between subsets of membrane-associated GH-binding sites, the serum GH-binding proteins and cytosolic GH-binding proteins dictates a reappraisal of earlier ligand-binding studies, which did not distinguish between binding-site subsets in the liver.

scholarly journals Soluble forms of the rabbit adipose tissue and liver growth hormone receptors are antigenically identical, but the integral membrane forms differ

1990 ◽  
Vol 267 (2) ◽  
pp. 471-477 ◽  
Author(s):  
R Barnard ◽  
S W Rowlinson ◽  
M J Waters
Keyword(s):  
Growth Hormone ◽  
Adipose Tissue ◽  
Binding Site ◽  
Hormone Receptors ◽  
Membrane Receptors ◽  
Liver Growth ◽  
Tissue Specific ◽  
Membrane Bound ◽  
Gh Receptors ◽  
Binding Subunit

Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for ‘somatogenic’ and ‘metabolic’ receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.

scholarly journals Evidence from the use of monoclonal antibody probes for structural heterogeneity of the growth hormone receptor

1985 ◽  
Vol 231 (2) ◽  
pp. 459-468 ◽  
Author(s):  
R Barnard ◽  
P G Bundesen ◽  
D B Rylatt ◽  
M J Waters
Keyword(s):  
Growth Hormone ◽  
Plasma Membrane ◽  
Binding Site ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Rabbit Liver ◽  
Receptor Subtypes ◽  
Hormone Binding ◽  
Gh Receptor

We describe the use of four monoclonal antibodies (MAbs) to the rabbit liver growth hormone (GH) receptor and one raised against purified rat liver GH receptor to characterize liver receptor subtypes which differ in their hormone-binding regions. The anti-(rat liver GH receptor) MAb both inhibited and precipitated rat and rabbit GH receptors, but only one-half of 125I-oGH (ovine GH) binding to liver microsomes could be inhibited by excess antibody. Conversely, only one-half of 125I-anti-(rat GH receptor) MAb binding was inhibited by excess oGH and Scatchard plots for this MAb exhibited two components. Although only 50% of 125I-oGH binding to membranes was inhibited by this MAb, all solubilized receptor could be immunoprecipitated. We postulate two epitopes for the anti-(rat GH receptor) MAb, one located at the hormone-binding site (inhibitory site) and one elsewhere (immunoprecipitating site). A second, rabbit-specific antibody (MAb 7) inhibited 85% of hormone binding but only 30% of 125I-anti-(rat GH receptor) MAb binding to rabbit liver microsomes. A combination of this MAb with the anti-(rat GH receptor) MAb totally inhibited 125I-oGH binding. MAb 7 alone totally inhibited 125I-rat GH binding to rabbit liver microsomes, as it did with 125I-oGH binding to purified receptor. On the basis of these results and others we postulate three types of GH receptor in rabbit liver membranes and ascribe approximate extents of 125I-oGH binding to each. A cytosolic ‘GH receptor’ which is not poly(ethylene glycol)-precipitable is shown to share five epitopes with ‘type 2’ microsomal receptors. Purified plasma membrane and endoplasmic reticulum fractions derived from a rabbit liver microsomal preparation have identical antigenic characteristics with respect to the GH-binding region, indicating that the heterogeneity we describe is not related to receptor processing. Of the three types of GH receptor in the plasma membrane of the rabbit (and possibly rat) we postulate that one (type 1) corresponds to the GH receptor involved in stimulating growth and possesses all of the epitopes studied here. A second (type 2) appears to be identical with the cytosolic ‘GH receptor’ and lacks the epitope for the anti-(rat GH receptor) MAb in the hormone binding site region. A third (type 3) does not possess the epitope for the inhibitory anti-(rabbit GH receptor) MAb, appears not to bind rat GH and is lost during purification. The availability of type-specific MAbs will facilitate assignment of specific functions to liver receptor subtypes which mediate the multiple functions of GH.

scholarly journals Influence of Mg2+ on detection of somatogenic and lactogenic components of growth-hormone-binding protein in mammalian sera

1993 ◽  
Vol 293 (2) ◽  
pp. 345-349 ◽  
Author(s):  
T Amit ◽  
Z Hochberg ◽  
R J Barkey
Keyword(s):  
Growth Hormone ◽  
Binding Protein ◽  
Rabbit Serum ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Type Iii ◽  
Wide Range ◽  
Human Sera ◽  
Serum Gh

We recently classified the growth-hormone (GH)-binding protein (GH-BP) in a wide range of mammalian [including human (h)] sera and reported the existence of a major lactogenic component in GH-BP of type-III sera (rabbit, horse, dog, pig and cat), based on the capacity of bovine (b) and ovine prolactin (PRL) to displace 125I-labelled human growth hormone (hGH) binding and on direct 125I-bPRL binding studies. In this study, we demonstrate the high degree of Mg2+ dependence of the binding of the classically lactogenic hGH and bPRL, but not that of the somatogenic bGH to various mammalian sera (types I-IV). Serum GH-BP was assayed using a previously described and validated charcoal-separation assay. 125I-hGH binding to rat, ovine, bovine, rabbit, horse, dog and human sera was enhanced 1.5-2.5-fold in the presence of 70 mM Mg2+. The Mg2+ effect was concentration-dependent between 3.7 mM and 70 mM, causing a significant and proportional increase in 125I-hGH binding to serum. Like 125I-hGH, 125I-bPRL binding to type-III sera was also Mg(2+)-dependent. In contrast, 125I-bGH binding to all types of serum GH-BP was not affected by Mg2+ concentrations of up to 35 mM, while 70 mM Mg2+ slightly, but significantly, reduced (by approx. 15%) bGH binding to rabbit serum. In keeping with the Mg(2+)-dependent stimulation of lactogenic hormone binding to GH-BP, 70 mM Mg2+ caused a shift to the left in the displacement curves of hGH and bPRL competing with 125I-hGH binding to rabbit, dog, horse and human sera, while the effects of the somatogens bGH and rabbit GH were shifted to the right. Scatchard analysis of hGH displacement curves with sera from various species yielded linear plots and revealed that Mg2+ significantly increased (2.3-3.0-fold) the affinity constants, but not the binding capacities. These results demonstrate the ability of changes in Mg2+ concentration to determine the degree of differential recognition of somatogens versus lactogens by serum GH-BP. It remains to be determined whether such bivalent cation effects may account, at least in part, for the growth retardation seen in Zn2+ or Mg2+ ion deficiencies.

Comparison between the human serum growth hormone-binding protein and the water-soluble growth hormone-binding site released from IM-9 lymphocytes

1993 ◽  
Vol 129 (6) ◽  
pp. 559-564 ◽  
Author(s):  
Guy Massa ◽  
Mapoko Ilondo ◽  
Magda Vanderschueren-Lodeweyckx
Keyword(s):  
Growth Hormone ◽  
Human Serum ◽  
Binding Site ◽  
Binding Protein ◽  
Water Soluble ◽  
Hormone Binding ◽  
Serum Growth Hormone ◽  
Gh Receptor ◽  
Growth Hormone Binding Protein ◽  
Hormone Binding Protein

The characteristics of the human serum growth hormone-binding protein (GHBP) were compared with those of a water-soluble GH-binding site prepared by incubating cultured IM-9 lymphocytes in assay buffer with 25 mmol/l iodoacetamide. High-performance liquid chromatography gel filtration of the water-soluble GH-binding site incubated with 125I-labeled human GH ([125I]hGH) revealed a large peak of bound [125I]hGH eluting at the same position as the peak of [125I]hGH bound to the GHBP in serum. The estimated Mr of the peak was 120 000, presumably representing one [125I]hGH bound to two binding sites. The binding specificities of the serum GHBP, the water-soluble GH-binding site and the GH receptor on IM-9 lymphocytes were identical. The binding affinities for 22 000 hGH and for 20 000 hGH of the serum GHBP were similar to the binding affinity of the water-soluble GH-binding site but lower than those of the cellular GH receptor. These findings show that the characteristics of the serum GHBP are comparable to those of the water-soluble GH-binding site released from IM-9 cells and support the hypothesis that in man the serum GHBP is produced by proteolytic cleavage of the cellular GH receptor.

Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit

1986 ◽  
Vol 881 (2) ◽  
pp. 236-240 ◽  
Author(s):  
Adrian C. Herington ◽  
Susie Ymer ◽  
Peter Roupas ◽  
Janet Stevenson
Keyword(s):  
Growth Hormone ◽  
High Speed ◽  
Binding Proteins ◽  
Hormone Binding

scholarly journals Multiple Growth Hormone-Binding Proteins are Expressed on Insulin-Producing Cells

1989 ◽  
Vol 3 (8) ◽  
pp. 1173-1182 ◽  
Author(s):  
Annette Møldrup ◽  
Nils Billestrup ◽  
Niels A. Thorn ◽  
Åke Lernmark ◽  
Jens Høiriis Nielsen
Keyword(s):  
Growth Hormone ◽  
Binding Proteins ◽  
Hormone Binding ◽  
Insulin Producing Cells
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