Identification of Two Specific Prolactin Binding Sites on Nb 2 Rat Lymphoma Cells

Abstract

Abstract

Several laboratories have earlier identified and characterized a specific prolactin binding site (receptor) on Nb₂ cells which has a Kd of about 2 × 10^-10 M and a binding capacity of about 12,000 receptor sites per cell. Using [l¹²⁵]-labeled human growth hormone (hGH), we have confirmed the results of these earlier reports. In addition, a second, “high-affinity” receptor site for prolactin has been identified. This site has a Kd of about 2 × 10^-12 M and there are an average of 67 sites per cell. Binding of [l¹²⁵]-hGH to the high-affinity receptor site is saturated by 1 min after incubation at 37°C, whereas saturation of the lower affinity site requires a 1-3 hr incubation at 37°C. Specificity of prolactin binding to both the high- and low-affinity “receptors” was established by the fact that several nonlactogenic hormones, including bovine growth hormone and insulin, do not displace [l¹²⁵]-hGH from the receptor sites. When Nb₂ cells were incubated for 1 hr at 0°C with 100 pg/ml [l¹²⁵]-hGH, no binding to the high-affinity receptor was detected, thus demonstrating the temperature dependence of binding to this receptor. The kinetics of binding of hGH to the high-affinity binding site correlates well with the dose-response effects of lactogenic hormones on mitogenesis in the Nb₂ cells. In addition, the rapid binding of hGH to the high-affinity receptor may be related to specific effects of lactogenic-hormones which occur within seconds or minutes after adding these hormones to Nb₂ cells.