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

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Abstract
The binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line was studied. The maximum bindability of radioiodinated 22,000- and 22,000- MW to IM-9 cells was 60 and 45%, respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10 h at 30.degree. 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 20,000 MW variant), -(1-134)- and -(141-191)-peptides. Both unlabeled hormones inhibit binding of both tracers completely, with the 20,000 MW variant being only half as potent as the 22,000 MW one. The apparent affinity is 2.8 .times. 109 M-1 for the 22,000 MW, variant and 1.6 .times. 109 M-1 for the 20,000 MW variant. This decreased affinity of the 20,000 MW variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the 2 variants that occupy .apprx. 15% of the total sites induce a marked down-regulation of the receptors after 18 h incubation, but the 20,000 MW variant (50% decrease) has a smaller effect than the 22,000 MW variant (75% decrease). The only consequence of the residues-32-46 deletion in the 20,000 MW variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the 2 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.