Cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rat GH receptor

Even though 20K human growth hormone (20K-hGH) has 3-10% binding affinity for the rat liver and adipose tissue microsomes as compared to 22K-hGH, it was also reported that 20K-hGH has the same potency as 22K-hGH in the hypophysectomized rat weight gain assay. In order to investigate the reason why such controversial data exist, we have studied 20K- and 22K-hGH using the rat GH receptor extracellular domain (rGHR-ECD) and full-length rGHR. When we examined the complex formation of rGHR-ECD with 20K- and 22K-hGH in gel filtration assay, 20K-hGH formed no complex while 22K-hGH formed a 1:1 complex. Next, rGHR cDNA was introduced into Ba/F3 cells and CHO-K1 cells, and stable transfectants (Ba/F3-rGHR and CHO-rGHR) were established. In the proliferation of Ba/F3-rGHR cells, 20K-hGH had 10-fold lower activity than 22K-hGH, which is consistent with their affinities for rGHR. But surprisingly, in the Spi2.1 gene promoter activation in CHO-rGHR cells, 20K- and 22K-hGH had the same activity, which was found not only in stable CHO-rGHR clones but also in CHO-K1 cells transiently expressing rGHR. In conclusion, these results indicate that cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rGHR.     

Use of calcium dependence as a means to study the interaction between growth hormones and their binding proteins in rabbit liver.

The affinity of 22,000-Mr human growth hormone (22 K-hGH) for GH binding proteins in rabbit liver is increased approx. 19-fold by 25 mM-Ca2+. In contrast, ovine growth hormone (oGH) binding is Ca2+-independent up to 10 mM, and decreased by greater Ca2+ concentrations. The 20,000-Mr hGH variant (20K-hGH), lacking residues 32-46, exhibits intermediate behaviour. Without Ca2+ there is a residual 40% of maximum specific binding to liver microsomes, and this increases to 65% with liver cytosolic GH binding proteins. In contrast with 22K-hGH, Scatchard analysis of 20K-hGH binding to liver microsomes produces curvilinear plots in the presence of 25 mM-Ca2+. From these results and inhibition studies with monoclonal antibodies to the GH binding proteins, it is concluded that deletion of the region 32-46 from 22K-hGH has eliminated one component of high-affinity Ca2+-potentiable binding. The Ca2+-mediated increase in Ka for the 22K-hGH-binding protein interaction is consistent with convergence of unit negative charges on the hormone and binding protein towards an intercalated Ca2+ ion. A positive charge in the critical region of nonprimate GHs would render their interactions Ca2+-independent and of lower Ka compared with 22K-hGH. A likely candidate for the negatively charged interactive residue is glutamate-33, since it is unique to human GH and is replaced by a positively charged arginine in non-primate GHs. Its absence in 20K-hGH could explain the altered calcium-dependence of 20K-hGH binding to what is probably the type 2 binding protein [Barnard & Waters (1986) Biochem. J. 237, 885-892]. The Ca2+-dependence of 20K-hGH binding to a subset of GH binding proteins provides both a verification and a mechanistic basis for the proposal [Hughes, Tokuhiro, Simpson & Friesen (1983) Endocrinology (Baltimore) 113, 1904-1906] that 20K-hGH binds with high affinity to only a subset of binding proteins in rabbit liver membranes.     

Demonstration of 20K growth hormone in human plasma by gel electrophoretic-immunostaining-autoradiographic assay (GEISAA)

A variant of human growth hormone (hGH), in which 15 amino acids are missing (commonly referred to as 20K-hGH in contrast to the traditional form which is referred to as 22K-hGH), is known to exist in human pituitary glands. However, lack of a method to measure it in blood has hindered investigations of its physiopathology. We have applied a newly-developed technique called GEISAA for its detection in small volumes of human plasma. The method is based upon the lower Mr of the variant and its ability to partially crossreact with existing antibodies for 22K-hGH. It consists of retrieval of the substance from plasma by immunoprecipitation, separation from 22K-hGH by NaDodSO4-polyacrylamide gel electrophoresis, transfer onto nitrocellulose paper by electroblotting and visualization by immunostaining and autoradiography. It revealed the 20K-hGH in plasma of some normal individuals and in that of an acromegalic patient. Furthermore, plasma concentration of the variant rose in conjunction with 22K-hGH following exercise, a natural stimulus for GH release. These results show that the 20K-hGH circulates under normal conditions and it is measurable by GEISAA using existing antibodies.     

High-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system

A chemically synthesized gene (hGHR-ED) coding for the extracellular domain (ED) of the human growth hormone (hGH) receptor (hGHR) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. The secreted 35-kDa protein was purified to near homogeneity. The purified protein exhibited a high binding affinity (Kd = 0.2-0.3 nM) to hGH. The highest cell production capability was estimated at more than 10-20 micrograms hGHR-ED/ml of culture. The inhibition of the hGHR-ED secretion by treatment with tunicamycin suggests that glycosylation is important for secretion.     

Structure of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor: evidence for strong structural coupling between receptor binding sites

The structure of the ternary complex between the phage display- optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 A resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH(v)) contains 15 Site 1 mutations and binds>10(2) tighter to the hGHR ECD (hGH(R1)) at Site 1. It is biologically active and specific to hGHR. The hGH(v) Site 1 interface is somewhat smaller and 20% more hydrophobic compared to the wild-type (wt) counterpart. Of the ten hormone-receptor H-bonds in the site, only one is the same as in the wt complex. Additionally, several regions of hGH(v) structure move up to 9A in forming the interface. The contacts between the C-terminal domains of two receptor ECDs (hGH(R1)- hGH(R2)) are conserved; however, the large changes in Site 1 appear to cause global changes in the domains of hGH(R1) that affect the hGH(v)-hGH(R2) interface indirectly. This coupling is manifested by large changes in the conformation of groups participating in the Site 2 interaction and results in a structure for the site that is reorganized extensively. The hGH(v)- hGH(R2) interface contains seven H-bonds, only one of which is found in the wt complex. Several groups on hGH(v) and hGH(R2) undergo conformational changes of up to 8 A. Asp116 of hGH(v) plays a central role in the reorganization of Site 2 by forming two new H-bonds to the side-chains of Trp104(R2) and Trp169(R2), which are the key binding determinants of the receptor. The fact that a different binding solution is possible for Site 2, where there were no mutations or binding selection pressures, indicates that the structural elements found in these molecules possess an inherent functional plasticity that enables them to bind to a wide variety of binding surfaces.     

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.     

The clinical usefulness of liquid human growth hormone (hGH) (Norditropin SimpleXx in the treatment of GH deficiency

Human growth hormone (hGH) is an essential therapeutic drug for the treatment of GH deficiency. The development of recombinant GH using a pen injection system has enabled easy and safe treatment of GH-deficient patients; however, the process of dissolving hGH in the powder form is complicated and dangerous. In this study, we investigated the usefulness of a newly developed liquid form of hGH (Norditropin((R)) SimpleXx(TM)) in the treatment of 51 patients with GH deficiency. Fifteen previously untreated patients with GH deficiency were treated with liquid hGH (group A), and 36 patients who had previously used hGH in the powder form were changed to the liquid form (group B). Both groups were treated with liquid hGH 0.5 IU/kg per week for 6 months. The growth rate of patients in group A increased from 4.0 +/- 2.4 cm/year to 9.2 +/- 2.9 cm/year. The patients in group B continued to grow at the same rate as before using the liquid hGH therapy. Questionnaires to the patients in group B demonstrated that 85% preferred the convenience of using the new liquid form of hGH. Our results indicate that liquid hGH has similar efficacy to that of powder hGH, but its improved convenience may have a beneficial effect on patient compliance.     

A comparison of subcutaneous and intramuscular administration of human growth hormone (hGH) and increased growth rate by daily injection of hGH in GH deficient children

Plasma human growth hormone (hGH) profiles and biological activities of recombinant hGH were compared after im and sc injection in 8 normal volunteers. The time to reach maximal plasma GH and plasma hGH concentrations and the areas under the curve of hGH profiles did not differ significantly after im and sc injections. The biological effect of hGH in increasing nonesterified fatty acid and insulin-like growth factor-I (IGF-I) was the same after both im and sc injections. During 6 months of daily sc administration of recombinant hGH in 20 naive patients, their height increased between 5 and 16.5 cm with a mean of 11.0 +/- 3.0 cm/year. In 27 patients who switched from hGH injections of 2-4 times/week to daily injections, the height increased between 5.3 and 16.5 cm with a mean of 8.3 +/- 2.2 cm/year. These values were greater than those observed in a previous study in which the same amount of hGH was injected in 2-4 doses per week. Plasma IGF-I increased more with daily sc administration than with 2-4 doses per week. The rate of appearance of an antibody to hGH was low (0.5%) and there were no notable changes in blood cell count, urinalysis and/or routine chemistries during the 6 months of daily recombinant hGH treatment. These results show that sc daily administration of hGH is safe, has a greater growth promoting effect, and can be recommended for the treatment of patients with GH deficiency.     

The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alanine-scanning mutagenesis: insights into the mechanisms responsible for improved affinity

A high-affinity variant of human growth hormone (hGH(v)) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) approximately 400-fold tighter than does wild-type (wt) hGH (hGH(wt)). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGH(v) binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGH(v) residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGH(v) differs significantly from that of hGH(wt). The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding "hot spot") remain important, but their contributions are attenuated in the hGH(v) interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGH(wt) are replaced by interactions that make positive contributions to the binding of hGH(v). These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone-receptor interfaces. Thus, it appears that the improved binding affinity of hGH(v) site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.     

IFN-gamma increases the hGH gene promoter activity in rat GH3 cells

AIM: To study the effect(s) of interferon gamma (IFN-gamma) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). METHODS: Cell transfection and luciferase reporter gene were used. RESULTS: IFN-gamma (10(2) and 10(3) U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 micromol/l) to the cells blocked the stimulatory effect of IFN-gamma. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-gamma induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-gamma, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-gamma was abolished following deletion of the -250 to -132 fragment. CONCLUSIONS: IFN-gamma increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-gamma appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-gamma requires the promoter sequence that spans the -250 to -132 fragment of the gene, but is unrelated to Pit-1 protein.     

Sensitive sandwich enzyme immunoassay of human growth hormone (hGH) in serum and urine using monoclonal antibody-coated polystyrene balls

A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGH IgG-coated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20 microliters of serum and 0.2 ng/l using 0.15 ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4 +/- 3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgG-coated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.     

Growth hormone (GH) stimulates protein synthesis in cells transfected with GH receptor complementary DNA

An expression vector containing a rat GH receptor cDNA was transfected into Chinese hamster ovary (CHO) cells, and stable cell lines expressing GH receptors were established. In contrast to nontransfected CHO cells, expression of GH receptors in transfected cells resulted in the appearance of high affinity (Kd = 1.53 nM) specific binding of GH. Cross-linking of [125I]hGH to the receptors and subsequent sodium dodecyl sulfate (SDS)-electrophoresis gave an estimated receptor mol wt of 84,000. GH treatment stimulated protein synthesis 60% over basal levels in GH receptor-expressing CHO cells, but not in the receptor-negative parental cells. The effect was observed only under serum-free conditions and was time and dose dependent. These results show that heterologous expression of the rat GH receptor results in the appearance of specific binding of GH and the acquisition of a functional GH response.     

Isolation of dimeric forms of human pituitary growth hormone

A procedure is described which for the first time allows the isolation of noncovalently-linked dimeric human pituitary growth hormone. Isomers of this dimeric species were prepared as were also, for the first time, isomers of covalently-linked dimers. Chromatography on DEAE-Sepharose CL-6B revealed the existence of noncovalently-linked dimers composed of monomers of 22K hGH, 20K hGH and 20K1 hGH (the latter is a new form of 20K hGH with a scission in the peptide chain) and covalent dimers containing 22K hGH and 24K hGH (the latter a 22K hGH with a scission). The different dimers all occurred as charge isomers and subsequent HPLC on an anion exchanger followed by zone electrophoresis in agarose suspension made possible the isolation of four noncovalently-linked isomers: one form of (20K-20K)hGH, two forms of (20K-22K)hGH and one form of (22K-22K)hGH; and of three covalently-linked isomers: one form of (22K-22K)hGH and two forms of (22K-24K)hGH.     

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.     

Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity

A chimeric bovine GH (amino acids Met-Asp-Gln-greater than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in Escherichia coli. The purified protein (chimeric GH) exhibited a 2-3 order of magnitude lower affinity toward lactogenic receptors in Nb2 lymphoma cells, microsomal fractions from bovine mammary gland and male rat liver. The affinity towards somatogenic receptors in IM-9 human lymphocytes and male rat liver was decreased to a much lesser degree. This diminished affinity towards lactogenic receptors was accompanied by a parallel decrease in the ability of the chimeric GH to stimulate proliferation of Nb2-11C lymphoma cells and the lipogenesis in bovine mammary gland. This implies that occupation of the respective receptors by either chimeric GH or hGH leads to identical postreceptoral effects. The chimeric GH was also capable of down-regulating the lactogenic receptors in Nb2 lymphoma cells and was recognized by three anti-hGH monoclonal antibodies. These and previously published results indicate that the N-terminal part of hGH is essential for the high affinity binding to lactogenic receptors and subsequent biological effect. Removal or replacement by a corresponding part of bovine GH converts the hormone, respectively to weak antagonist or agonists. Analysis of our data, based on hydropathy index leads us to suggest that the high affinity binding site of the hGH towards lactogenic receptors is mainly confined to amino acids nos. 8-18.     

Lipolytic and antilipolytic effects of human growth hormone, its 20-kilodalton variant, a reduced and carboxymethylated derivative, and human placental lactogen on chicken adipose tissue in vitro

The lipolytic and antilipolytic effects of human growth hormone (22K-hGH), its 20-kilodalton variant (20K-hGH), a reduced and S-carboxymethylated derivative (RCM-hGH), and human placental lactogen were examined using chicken adipose tissue explants in vitro. Lipolysis, as determined by glycerol release, was stimulated by 22K-hGH (biosynthetic and pituitary derived), 20K-hGH (pituitary derived), and RCM-hGH (modified biosynthetic). These growth hormone preparations also exhibited similar antilipolytic activity (i.e., transient inhibition of glucagon-induced lipolysis). However, unlike human growth hormone, human placental lactogen neither stimulated lipolysis nor inhibited glucagon-stimulated lipolysis. Some augmentation of glucagon-stimulated lipolysis was observed in the presence of human placental lactogen. These results indicate that the disulfide bridges (Cys53----Cys165; Cys182----Cys189) and amino acid residues 32-46 of hGH are not required for lipolytic or antilipolytic activities of human growth hormone on chicken adipose tissue.     

Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core

A 36-kDa protein, which is a component of the membrane skeleton, has been shown to co-localize with spectrin in addition to serving as a major substrate for tyrosine-protein kinases. This protein, which will be referred to as calpactin (for calcium-dependent phospholipid and actin binding protein), was isolated from bovine intestine as the complex with a 10-kilodalton light chain and the Ca2+ binding was analyzed by equilibrium dialysis with 45Ca2+ in the presence or absence of phospholipid. Although Ca2+ binding by calpactin alone was negligible at micromolar free Ca2+, it was greatly enhanced by liposomes containing phosphatidylserine or phosphatidylinositol. A proteolytic derivative of calpactin, termed the "core," which has lost the site of association with the light chain in addition to the site of tyrosine phosphorylation by pp60src, was also found to contain this high affinity phospholipid enhanced Ca2+-binding activity. Scatchard plots reveal that each calpactin monomer or core polypeptide bound 2 Ca2+ ions with a Kd of 4.5 X 10(-6) M at 200 micrograms of phosphatidylserine/ml. Liposome binding experiments confirmed that calpactin as a complex with light chain as well as calpactin monomer or the 33-kDa core interact with phosphatidylserine liposomes in a Ca2+-dependent manner.