The binding of rat liver cell multiplication-stimulating activity (MSA) to human placenta and serum proteins.

Multiplication-stimulation activity (MSA) from the medium of BRL-3A rat liver cells in culture binds to cell membrane and cytosol receptors from human placenta and to serum proteins. The binding of MSA to placental cell membranes is dependent on time, temperature, pH and divalent ion concentration. MSA bound to placental cytosol receptor and serum is not displaced by insulin, whereas that bound to placental cell membranes is displaced by insulin and insulin-like peptides. The affinity of the three receptors for MSA is similar [approximately 10(8) M(-1)]. An assay using 125I-MSA and placental membrane receptor detects somatomedin-like receoptor activity (SmLRA) in unextracted sera from man and animals. A binding protein in serum that competes for 125I-MSA with receptor could not be completely separated from SmLRA by heating, acidification, charcoal treatment and gel chromatography of the serum. The relative activities of SmLRA and serum binding protein remained constant in three disorders of human growth (acromegaly, growth hormone deficiency and Laron's dwarfism) in which values of SmLRA varied widely. However, the binding protein is only partly responsible for the apparent SmLRA of unextracted serum. It is concluded that MSA is a suitable radioligand for the investigation of somatomedin disorders in man either by receptor assays or by studies of tissue receptors.     

Regulation of GH binding to specific cellular receptors in vitro--a new model of growth regulation in vivo

A new theory on the complex growth hormone (GH) mediated promotion of tissue growth has been developed on the basis of in vitro studies of growth hormone receptors on human peripheral mononuclear cells (PMC). It is hypothesized that GH acts through its tissue receptors and regulates its own receptors through two different pathways: first GH directly downregulates GH binding, second a partially GH-dependent serum factor (the so-called SM-B) enhances GH binding. Some experimental evidence for this hypothesis is presented using a new method to investigate GH receptors in circulating human blood cells: The effect of trypsin, antitrypsin, growth hormone, somatomedin-B (SM-B) and anti-SM-B-antiserum on GH binding to PMC was studied. Trypsinization of cells leads to a decrease both of specific binding and of binding affinity (affinity constant after 60 minutes of trypsinization 0.5 X 10(6) M-1 versus 1.5 X 10(6) M-1 in untreated control cells). Exposure of PMC to antitrypsin activities was followed by an increase of binding affinity and specific binding (affinity constants with 10 KIU 1.9 X 10(6) M-1, with 100 KIU 2.4 X 10(6) M-1, with 1000 KIU 3.6 X 10(6) M-1). This antitrypsin effect exceeds the binding values expected after blocking trypsin activities possibly being present in the incubation medium. In a subset of experiments the partially GH-dependent serum factor SM-B was used as the antitrypsin moiety and was shown to increase specific GH binding to PMC in a similar manner as did antitrypsin (with 1000 ng SM-B affinity constant 12.0 X 10(6) M-1, specific binding 9.7%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.     

Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy

The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).     

Receptor of prolactin and its effect on binding of corticotropin in human and guinea pig adrenocortical cells

Specific binding of prolactin (PRL) by human and guinea pig isolated adrenocortical cells is saturable and reach equilibrium during 60 min. Characteristics of PRL binding by adrenocortical microsomes were determined. A single receptor species related to the high affinity and low binding capacity receptor class was revealed. The PRL-receptor affinities in adrenal cortex and liver are similar (approximately 10(9) M-1). Human adrenals have a higher affinity than that in guinea pigs. PRL increased corticotropin binding by adrenocortical cells of both species. Corticotropin binding rise may be due to the increase in the number of available receptors of low affinity and high capacity.     

Kinetic analyses of the binding of leukemia inhibitory factor to receptor on cells and membranes and in detergent solution.

The equilibrium and kinetic properties of leukemia inhibitory factor (LIF) binding to a range of cell types have been compared. When binding was examined at 4 degrees C, the majority of cells were found to express a single class of high affinity LIF receptor (KD = 20-100 pM; ka = 2-8 x 10(8) min-1 M-1; kd = 0.0004-0.0011 min-1). In contrast, certain activated macrophage populations expressed apparently independent classes of high and low affinity LIF receptor. The low affinity receptors differed from the high affinity receptors in terms of the dissociation rate of the receptor-ligand complex (KD = 1-2 nM; ka = 3-7 x 10(8) min-1 M-1; kd = 0.30-0.67 min-1). At 37 degrees C, the interaction of LIF with its high affinity receptor was more complicated, since occupied LIF receptors were internalized more rapidly than unoccupied receptors, internalized LIF was hydrolyzed and released from the cell, and new receptors were synthesized and expressed on the cell surface. Interestingly, when membranes were prepared from cells that expressed only high affinity receptors, both high and low affinity receptors were detected, while after detergent solubilization of membranes only low affinity receptors were apparent. These results are discussed in terms of a structural model for the LIF receptor in which interaction of a low affinity binding subunit and a second nonbinding subunit is required for the generation of the high affinity receptor.     

Molecular mechanisms of olfactory reception. VI. Kinetic characteristics of camphor interaction with binding sites of rat olfactory epithelium

Camphor binding to a possible receptor of rat olfactory epithelium has been studied within the ligand concentration range 10(-11)-10(-6) M. At these concentrations camphor is bound by a set of receptors. They are distinguished by both the affinity to the ligand (K1 = 5 X 10(-10) M, K2 = 3.5 X 10(-8) M, K3 approximately equal to 10(-6) M) and their amount in the epithelium. The differences in the affinities are due to different values of the association rate constant of camphor (k1), which varies from 10(6) M-1 X s-1 for the receptors with high affinity up to 2 X 10(2) M-1 X s-1 for those with low affinity. These data are discussed in terms of equilibrium and kinetic models of the receptor-stimulus interaction.     

Protein binding to brush borders of enterocytes from the jejunum of the neonatal rat

The specific binding of IgG to jejunal brush borders was greatest at acidic pH, at neutral pH no specific binding occurred. Specific binding declined with age-no specific binding occurred in borders from 20-and 24-day-old animals. There was no specific binding of IgG to borders from ileal enterocytes. Human transferrin and bovine serum albumin did not bind specifically to borders. The affinity of binding (-Ka) and the receptors site numbers per border estimated for rat IgG were 18.64 X 10(6) M-1 to 3.53 X 10(6) sites; for human IgG, 25.06 X 10(6) M-1 to 3.30 X 10(6) sites; for bovine IgG, 10.48 X 10(6) M-1 to 2.11 X 10(6) sites and for sheep IgG, 7.26 X 10(6) M-1 to 2.34 X 10(6) sites.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Insulin binding on cultured chick muscle cells: decrease in binding associated with cell fusion

Binding of 125I-bovine and chicken insulin to cultured embryonic chick skeletal muscle cells was studied. Bovine and chicken insulin bound cultured cells with high affinities of 2.4 X 10(9)M-1 and 4.8 X 10(9)M-1 and low affinities of 2.4 X 10(7)M-1 and 3.7 X 10(7)M-1, respectively. Maximum insulin binding was achieved after 90 min of incubation at 20 degrees C and the maximum value was maintained for an additional 3 hr. Insulin binding increased in a linear manner with increasing nuclei number over a 5-fold range. Maximum insulin binding per nuclei decreased as cell fusion increased between 24 and 72 hr in culture, primarily due to a decrease in the number of low affinity insulin receptors.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.     

Characterization of prolactin receptors in pig mammary gland.

Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

Receptor-binding and down-regulatory properties of 22000-Mr human growth hormone and its natural 20000-Mr variant on IM-9 human lymphocytes.

Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.