Experimental Biology and Medicine

Concurrent studies conducted in several laboratories in the late 1980s led to cloning, expression, and characterization of the receptors to growth hormone (GH), prolactin (PRL), erythropoietin (EPO), interleukin (IL)-4, IL-7, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In 1989-90 these were recognized as a novel family of growth factor receptors, followed by an explosion of publication in the field and extension of the family to include several other growth factors, mostly cytokines. These receptors were found to have a high degree of homology in sequence motifs of their N-terminal extracellular domains. These extracellular domains were the subject of another important pursuit by investigators: recognizing soluble binding proteins for several of the ligands to the superfamily, with sequence identity to the extracellular domains of the receptors.

Cell viability, multiplication, and differentiation to the various hematopoietic cell lineages are induced by a multigene cytokine family, and hematopoiesis is controlled by a network of interactions between these cytokines. This network includes positive regulators such as colony-stimulating factors and interleukins, and negative regulators such as transforming growth factor-β and tumor necrosis factor. The functioning of the network requires an appropriate balance between positive and negative regulators, and the selective regulation of programed cell death (apoptosis) by interaction of cytokines with their receptors. The cytokine network, which has arisen during evolution, allows considerable flexibility, depending on which part of the network is activated, and the ready amplification of response to a particular stimulus. This amplification occurs by autoregulation and transregulation of genes for the hematopoietic cytokines. There is also a transregulation by these cytokines of cytokine receptors. In addition to the flexibility of this network, both for response to present day infections and to infections that may develop in the future, a network may also be necessary to stabilize the whole system. The existence of a network and the cytokine-receptor regulation of apoptosis has to be taken into account in the clinical use of cytokines for therapy. Cytokines that regulate hematopoiesis induce the expression of genes for transcription factors. Cytokine signaling through transcription factors can thus ensure the autoregulation and transregulation of cytokine and receptor genes that occur in the network. Interactions between the cytokine network and transcription factors can also ensure production of specific cell types and stability of the differentiated State.

The strength of the cellular immune response is regulated to a large extent by the amount of interleukin-2 (IL-2) produced in response to a stimulus. The ability of lymphocytes and other cells to respond to IL-2 depends upon the expression of cell surface IL-2 receptors. Formation of a high-affinity IL-2 receptor is regulated primarily through induction of its a subunit, IL-2Rα. Once formed, the IL-2Rα chain turns over rapidly, rendering expression of high-affinity IL-2 receptors during the immune response dependent upon continuous activity of the IL-2Rα gene. The induced expression of both human IL-2 and IL-2Rα chains is sensitive to cell-mediated suppression by CD8 cells; depletion of CD8 cells leads to extensive superinduction. This coupled suppression of IL-2 and IL-2Rα genes greatly increases the extent of control, and strongly limits the strength, of the signal transduced by this ligand/receptor system during an immune response.

The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determine pathways whereby GH signals are conveyed within the cell.

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHR_l) and a smaller isoform, GHR_s that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHR_s may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHR_s was ligated into the mammalian expression vector pcDNAl/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHR_s in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHR_l in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHR_l. The transfected cDNA for GHR_s directs the synthesis of a 50 kDa protein. Cross-linking of [¹²⁵l]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHR_s[¹²⁵l]hGH complex. In the presence of 10% newborn calf serum, incorporation of [³⁵S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by ~70% in control cells, but in the transfected cells, protein synthesis was reduced only by ~50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although ³H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by ~70% in control cells, but only by ~30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr of serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHR_s produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHR_s. We conclude that GHR_s contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHR_s in 3T3-L1 cells altered cell function in the absence of GH.

Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb₂ cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo.

Growth hormone receptor (GHR) expression has been analyzed at the RNA level. In the rat, relative expression of the RNA species encoding the GHR and the GH-binding protein (GHBP) appears to be sensitive to endocrine status. Full-length GHR cDNA clones from ovine, porcine, and chicken were used as probes to investigate the existence of unique RNAs for GHBPs in these species. In the sheep and pig, only a single, ~4.5-kb RNA is apparent. Although quite high levels of GH binding activity are found in pig serum, a variety of methods failed to isolate a separate GHBP message, suggesting that porcine GHBP is produced via a mechanism different from that which is known for rat. One class of chicken GHR cDNA, resulting from alternative use of a splice acceptor 17 bases upstream of the Intron 6/Exon 7 junction, is also presented.

Despite extensive studies, the structure of the erythropoietin receptor remains little understood. cDNAs encoding the human and murine erythropoietin receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in erythropoietin binding and in erythropoietin signal transduction, increasing evidence strongly suggests that the erythropoietin receptor is a multimeric complex. The murine erythropoietin receptor has been solubilized under mild conditions and the molecular mass of the native receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other erythropoietin receptor subunits.

The growth hormone (GH) receptor belongs to the GH/prolactin/cytokine super-family of receptors. The signal transduction mechanism utilized by this class of receptors remains largely unknown. In order to identify functional domains in the intracellular region of the GH receptor we generated a number of GH receptor mutants and analyzed their function after transfection into various cell lines. A truncated GH receptor missing 184 amino acids at the C-terminus was unable to mediate GH effects on transcription of the Spi 2.1 and insulin genes. However, this mutant was fully active in mediating GH-stimulated metabolic effects such as protein synthesis and lipolysis. Furthermore, this mutant GH receptor internalized rapidly following GH binding. Another truncated GH receptor lacking all but five amino acids of the cytoplasmic domain could not mediate any effects of GH nor did it internalize. Deletion of the prolinerich region or changing the four prolines to alanines also resulted in a GH receptor deficient in signaling. Mutation of phenylalanine 346 to alanine resulted in a GH receptor which did not internalize rapidly; however, this mutant GH receptor was capable of mediating GH-stimulated transcription as well as metabolic effects. These results indicate that the intracellular part of the GH receptor can be divided into at least three functional domains: (i) for transcriptional activity, two domains are involved, one located in the C-terminal 184 amino acids and the other in the prolinerich domain; (ii) for metabolic effects, a domain located in or near the proline-rich region is of importance; and (iii) for internalization, phenylalanine 346 is necessary.

The intracellular pathways by which the binding of growth hormone (GH) to its receptor elicits its diverse effects have eluded investigators for many years. Studies showing that GH rapidly stimulates tyrosyl phosphorylation of cellular proteins, and that tyrosine kinase activity co-purifies with GH-GH receptor complexes, led us to hypothesize that activation by GH of a receptor-associated tyrosine kinase is an important early, and perhaps, initiating step in signal transduction by GH. Here, we review the work identifying JAK2 as a GH receptor-associated tyrosine kinase that is rapidly activated by ligand binding.

It has been proposed that dimerization of identical receptor subunits by growth hormone (GH) is the mechanism of signal transduction across the cell membrane. We present here data with analogs of porcine GH (pGH), with GH receptors (GHR) mutated in the dimerization domain and with monoclonal antibodies to the GHR which indicate that dimerization is necessary but not sufficient for transduction. We also report nuclear uptake of GH both in vivo and in vitro, along with nuclear localization of the receptor and GH-binding protein (GHBP). This suggests that GH acts directly at the nucleus, and one possible target for this action is a rapid increase in transcription of C/EBP delta seen in 3T3-F442A cells in response to GH. This tyrosine kinase-dependent event may be an archetype for induction of other immediate early gene transcription factors which then interact to determine the programing of the subsequent transcriptional response to GH.

The Neu proto-oncogene (also called ErbB-2 and HER-2) encodes a tyrosine kinase transmembrane receptor homologous to the epidermal growth factor receptor (EGF-R). Overexpression, a point-mutation, and co-expression with EGF-R activate the oncogenic potential of the Neu protein by permanent coupling to signal transducing pathways. The search for ligands that elevate tyrosine phosphorylation of Neu led to the discovery of a 44-kDa glycoprotein that acts either as a differentiation factor or as a mitogen for mammary tumor cells. This protein, termed Neu differentiation factor (NDF), is derived from a transmembrane precursor that contains an EGF-like motif and an immunoglobulin-like domain. Alternative splicing generates a dozen NDF-related proteins that are expressed in a variety of mesenchymal and neuronal tissues. This unprecedented multiplicity raises the possibility that different isoforms fulfill distinct biological roles.

Interferons (IFNs) act by inducing several intracellular antiviral proteins. We report here that IFNs also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This protein accounts for 25%-50% of the total antiviral activity elicited by IFN. The antiviral protein was purified to homogeneity from culture supernatants of IFN-treated cells by several chromatographic steps, to give a single 28-kDa active polypeptide. Upon sequencing, this novel protein corresponded to the N-terminal ligand-binding domain of the human 160-kDa low-density lipoprotein receptor (LDLR). In addition, we find that IFN induces the cell surface LDLR and this phenomenon may explain previous reports on reduction of serum cholesterol in IFN-treated patients. Viruses produce soluble cytokine receptors that inhibit their respective cytokines, thereby assisting virus infection. It appears now that host cells employ similar molecules for the opposite role of controlling virus infections.

We investigated the role of soluble interleukin-4 receptor (slL-4R) as a regulator of IL-4 mediated activities in vivo. Administration of recombinant sIL-4R to mice resulted in (i) prolonged survival of heterotopic cardiac allografts; (ii) decreased popliteal lymph node enlargement in response to allogeneic cells; and (iii) inhibition of IgE secretion in response to anti-lgD treatment. Transgenic mice constitutively expressing elevated levels of biologically active SIL-4R displayed prolonged cardiac allograft survival compared with control animals. However the SIL-4R transgenic mice were capable of mounting normal antigen-specific IgE responses despite the presence in serum of up to 3 μg/ml slL-4R. Surprisingly, coadministration of IL-4/slL-4R or IL-4/anti-IL-4 mAb complexes caused a superinduction of IgE secretion in anti-lgD-treated normal mice and subsequently in other IL-4-dependent biological activities. Thus, recombinant slL-4R can not only antagonize functions mediated by endogenous IL-4, but also potentiate the biological activity of exogenously administered IL-4. These dual roles may have possible clinical implications for the recombinant molecule, as well as for natural slL-4R immunoregulation.

Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

The direct role of prolactin (PRL) in testicular function is still unclear, mostly because of lack of a suitable in vitro model. To establish the suitability of the MA-10 murine tumor Leydig cell line for the study of PRL receptors (PRLR) and effects on steroidogenesis, we initially characterized PRLR on cultured MA-10 cells. The specific binding (B_s) of [¹²⁵l]human growth hormone (hGH) depends on time, temperature, and Mg²⁺ ion and protein concentrations, with absolute specificity for the lactogenic hormones hGH and ovine PRL. B_s is saturable and is to a single class of high-affinity (K_a = 3.6 × 10⁹ M ^-1) low-capacity (B _max = 19.5 fmol/mg protein) binding sites. The molecular weight of PRLR, determined by cross-linking to [¹²⁵l]hGH, SDS-PAGE and autoradiography, is 35 kDa for the free receptor, suggesting that the short-form PRLR protein, previously described in liver and mammary glands, is that primarily found in MA-10 cells. Thus, the demonstration of specific PRL binding sites on MA-10 Leydig cells, with characteristics similar to primary Leydig cell PRLR, suggests that this cell line can serve as a good model for both the study of PRLR mechanism of action and the role of PRL in Leydig cell function.

The pulsatile pattern of growth hormone (GH) secretion is synchronized with the GH-receptor (GHR) turnover and the ensuing GH-binding protein (GHBP). We investigated the effect of GH pulse frequency on the turnover of GHR, and tested the theoretical reciprocal impacts of GH, GHR, and GHBP in different species and clinical conditions. Male Sprague-Dawley rats were hypophysectomized (hypox) at 35 days of age. Two groups of 16 rats received a single or two iv injections of 10 μg human GH (hGH) at an interval of 45 min. They were killed 45, 90, or 135 min after the single or second injection. A third group of 25 hypox rats were given continuous sc infusion of hGH for 6 days. Liver membranes were prepared for hGH somatogenic binding. A bolus of GH at 45-min intervals expedited GHR turnover, and continuous GH resulted in faster turnover cycles of 90 min. The impact of GHBP on GH bioactivity was then calculated in human serum by a rabbit liver membrane displacement assay. Bioactivity was diminished by GHBP with increasing GH levels up to a point, within the physiological range, where GH bioactivity is gradually restored. Finally, simulation calculation of the bound and free fraction of GH over a typical pulse in man and male rat showed the changing relations of free/bound GH. In man free hormone predominates most of the pulse, whereas in rat free GH comprised a smaller fraction. Thus, the reciprocal effects of GH on GHR turnover and GHBP generation, and that of GHBP on GH t _1/2 lead to self-perpetuation of high (mostly free) GH in downregulation of GHR and its turnover with resultant lower GHBP.

The mouse growth hormone receptor (mGHR) and the mouse growth hormone-binding protein (mGHBP) are products of a single gene which are generated by alternative splicing. The factors that regulate the expression of mGHR and mGHBP mRNA and protein during pregnancy in the mouse are incompletely understood. During pregnancy in the mouse, there are parallel increases in circulating mouse growth hormone (mGH), liver mGHR, and serum mGHBP. The increase in both hepatic mGHR and serum mGHBP begins on Day 9 of gestation and by late gestation the hepatic mGHR content has increased 8-fold and serum mGHBP has increased 30-fold compared with values in nonpregnant controls. A parallel increase occurs in the steady state levels of liver GHR and GHBP encoding mRNAs. The increase in both messages begins on Day 9 of gestation; however, the GHR mRNA reaches maximum levels by Day 13, while the GHBP mRNA continues to increase until the end of pregnancy. The magnitude of the increase in the GHR-encoding message is 15- to 20-fold between nonpregnant and late pregnant mice, and the magnitude of the increase in the GHBP-encoding message is 30- to 50-fold. Both pituitary mGH and the number of conceptuses influence the receptors and binding protein for mGH during pregnancy.

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR.

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free calcium concentration ([Ca₁]) in single human BFU-E derived erythroblasts, which is specific for stage of differentiation and mediated through a voltage-independent ion channel permeable to calcium. This minireview examines the regulation of calcium channels by Epo during normal erythroid differentiation, as a model to delineate the immediate signaling events which follow interaction of Epo with its receptor.

Many cytokines initiate cellular responses through their interaction with members of the cytokine receptor superfamily which contain no catalytic domains in their cytoplasmic domains. Irrespective, ligand binding induces tyrosine phosphorylation, which requires a membrane proximal region of the cytoplasmic domain. Recent studies have shown that members of the Janus kinase (JAK) family of protein tyrosine kinases associate with the membrane proximal region, are rapidly tyrosine phosphorylated following ligand binding and their in vitro kinase activity is activated. The JAKs are 130-kDa proteins which lack SH2/SH3 domains and contain two kinase domains, an active domain and a second kinase-like domain. Individual receptors associate with, or require, one or more of the three known family members including JAK1, JAK2, and tyk2. Substrates of the JAKs include the 91-kDa and 113-kDa proteins of the interferon-stimulated transcription complex ISGF3. These proteins, when tyrosine phosphorylated, migrate to the nucleus and participate in the activation of gene transcription. Recent evidence suggests that the 91- and 113-kDa proteins are members of a large family of genes that are potential substrates of JAK family members and may regulate a variety of genes involved in cell growth, differentiation or function.

Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E. coli (amino acids 4-210) expression systems. Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E. coli expression system. All four proteins were purified as monomers with >95% homogeneity. Their affinity for various lactogenic and somatogenic hormones was determined by binding assays. The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays. The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity. This conclusion does not preclude however, hormoneor antibody-induced dimerization of the membrane-embedded receptor.

Prolactin (PRL) and growth hormone (GH) receptors are members of a superfamily that include receptors for a number of cytokines. GH and its receptor form an unusual homodimer consisting of one molecule of GH and two molecules of receptor. A similar homodimer of the PRL receptor is probably required for biological effects to be seen. Using specific assays to measure the functional activity of PRL and GH receptors, a 25 amino acid juxtamembrane region has been identified as essential but not sufficient for normal action. More detailed studies have limited the region to eight amino acids, rich in prolines, that is highly conserved in many members of the receptor superfamily. Finally, GH and PRL have been shown to induce the rapid tyrosine phosphorylation of an associated kinase, Janus kinase 2, and of the receptor itself.

The cytokines, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), share many similar activities on cells of the hemopoietic system. Cloning of the receptors for IL-3 and GM-CSF revealed that these two cytokines utilize receptors consisting of a ligand-specific α unit and a β subunit which is shared. Neither subunit contains intrinsic tyrosine kinase activity, but deletion analysis of the β subunit cytoplasmic domain has defined certain regions which are important for signal transduction.

Ectopic expression of the erythropoletin receptor (Epo-R) in Ba/F3, an interleukin-3 (IL-3)-dependent progenitor cell line, confers both Epo-dependent cell growth and Epo-dependent induction of β-globin mRNA. We have used this system of limited erythroid differentiation to characterize the role of the Epo-R in differentiation. In particular, we have been interested in identifying a differentiation domain of the Epo-R. We have studied three chimeras encoding regions of the extracellular region of the Epo-R and the intracellular region of the IL-3Rβ_IL3. After transfection into Ba/F3 cells, all three chimeras conferred Epo-dependent growth and induced the expression of β-globin, suggesting that the extracellular region of the Epo-R plays a critical role in differentiation. However, a truncated Epo-R containing only the extracellular region of the Epo-R and a 15 amino acid cytoplasmic tall does not induce β-globin expression, although it is processed to the cell surface and binds Epo. These experiments show that the extracytoplasmic region of the Epo-R is necessary but not sufficient to induce erythroid-specific differentiation in this system.

The complex interaction of leukemia inhibitory factor (LIF) with its specific receptor present on the cell surface, in isolated membranes and in solution, has been examined in detail. Several aspects of this complexity have been highlighted, including the presence of high- and low-affinity murine LIF receptors, biphasic dissociation of human LIF from apparently homogeneous high- or low-affinity human LIF receptors, and unusual species cross-reactivity. The unusual species cross-reactivity observed between murine and human LIF has also been exploited to map an important receptor binding epitope on human LIF.

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca⁺⁺ concentration. PRL stimulates Ca⁺⁺ entry and induces secondary Ca⁺⁺ mobilization. The entry of Ca⁺⁺ is a result of an increase in K⁺ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 μM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the β lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

An isoform of the growth hormone (GH) receptor that lacks the transmembrane and intracellular domains is formed in adipocytes by alternate splicing of mRNA. This isoform is identical to the circulating GH-binding protein. The short isoform is about six times as abundant as the long isoform in rat adipocytes. It is located largely in the cytosolic compartment in association with intracellular membranes, but about 20% is present on the adipocyte surface as judged by the susceptibility to digestion by trypsin. In contrast, about 80% of the long isoform of the receptor is present on the cell surface. The two isoforms turn over at different rates and appear to be independently regulated. Both appear to contribute equally to GH binding. In preliminary studies, immunoneutralization of the short isoform decreased the magnitude of the effect of GH on glucose metabolism, suggesting that the short isoform may mediate some of the responses to GH.

The high-affinity interleukin-2 receptor (IL-2R) is a multichain receptor with at least three IL-2 binding chains: IL-2Rα (55 kDa) bound by the monoclonal antibody anti-Tac, IL-2Rβ (75 kDa) and IL-2rγ (64 kDa). The IL-2Rα also exists as a naturally occurring soluble molecule (sIL-2Rα). We target the IL-2R for immune intervention since resting normal cells do not express the high-affinity IL-2R, whereas this receptor is on some cells in certain lymphoid neoplasias, select autoimmune disorders, and in individuals rejecting organ allografts. Treatments have included unmodified murine anti-Tac and radioisotopes conjugated to murine anti-Tac. Our emerging understanding of the IL-2R system continues to open possibilities for more specific immune intervention.

The first method used for detection of growth hormone-binding protein (GHBP) in biological fluids was based on the incubation of the sample with radiolabeled GH followed by separation of bound and free GH by gel exclusion chromatography. Recently, other methods have been developed which are faster and easier to use. These methods include variants of the original binding/column assay (e.g., separation of bound and free GH is obtained by immunoprecipitation, charcoal adsorption, ion exchange chromatography, or HPLC), and a ligand-mediated immunofunctional assay (LIFA), in which a monoclonal antibody is used to capture the GHBP on a microtiter plate; all binding sites are saturated with GH and an anti-GH antibody is used to detect the amount of GH (endogenous and exogenous) bound to the GHBP. To permit comparison of results obtained by different methods we have cross-validated the LIFA with two different binding assays: (i) the original long column assay (column assay), and (ii) an assay based on immunoprecipitation (RIPA) of the GH/GHBP complex with an anti-GHBP antibody.

Serum levels of growth hormone-binding protein (GHBP) were measured by high-performance liquid chromatography (HPLC) gel filtration in serum of 30 pregnant women and in cord serum of 69 preterm and term infants. In pregnant women, the mean ± SD serum level of GHBP was 32.4% ± 5.6%. Polynomial regression analysis showed a significant (P = 0.01) second-order relationship between the duration of pregnancy and serum GHBP levels, with increasing levels during the first half of pregnancy and decreasing levels thereafter. In cord serum, the mean ± SD serum level of GHBP was 3.1% ± 1.4% (n = 51) in preterm and 4.2% ± 1.3% (n = 18) in term neonates. Serum GHBP concentrations were related to gestational age (r = 0.35; P < 0.005) and to intrauterine nutritional state as evaluated by the ponderal index (r = 0.30; P < 0.02). In four term neonates, serum levels of GHBP were measured before and after an exchange transfusion (ET). Before the ET the serum GHBP level was 6.7% ± 2.4% and at the end it was 25.3% ± 2.6%. Thereafter, serum GHBP levels decreased slowly. Analysis of the elimination curve by exponential stripping showed a biexponential elimination pattern: GHBP (%) = 40· e(^{-5.1E-03 × time)} + 64 · e(^{-2.5E-04 × time)}. The serum half-life of the GHBP complex was estimated to be about 2 days.

To investigate the role of growth hormone (GH) and its downstream axis in normal growth and growth disorders, we measured serum GH-binding protein (GHBP) levels in children by ligand-mediated immunofunctional assay (UFA). Samples were taken from 512 healthy children of normal stature, 146 healthy neonates, 153 short but otherwise normal children, and 27 patients with GH deficiency. Serum GHBP showed no significant diurnal variation. Serum GHBP levels were low in neonates, acutely rose within 6 months, and gradually increased toward midpuberty in normal children (r = 0.2, P < 0.01, assessed among subjects over 1 year old). Neither significant sexual dimorphism nor rapid pubertal changes were observed. In normal short children and patients with GH deficiency, GHBP was lower than normal, but not significant. These data suggest that one of the etiologies for growth disorder in children could be resistance to, or low efficacy for, utilization of GH.

Three Laron Syndrome (LS) siblings with a post growth hormone (GH) receptor defect for insulin-like growth factor-l (IGF-I) synthesis were found to have serum GH-binding protein (GHBP) levels normal for age. Treatment with recombinant IGF-I (150 μg/kg/day) decreased serum GHBP activity to 62% of the basal value (P < 0.001) in two of the sibs in 1 week and in the third sib after 3 months of therapy. Scatchard analysis of the binding of [¹²⁵I]human GH (hGH) to GHBP in patients' sera before and during therapy revealed affinity constants K_a = 1.55-1.80 × 10⁹ M ^-1, similar to that of sera from healthy subjects. Variations in binding are due to changes in the binding capacity. IGF-I may be a regulatory factor for serum GHBP activity in man.