Direct visualization of membrane clustering and endocytosis of thyrotropin into cultured thyroid cells

We prepared a highly fluorescent conjugate of thyrotropin (TSH) which retains approximately 25% of the binding affinity of native TSH towards TSH receptor and approximately 25% of the potency of the native hormone in stimulating the accumulation of cAMP in thyroid cells. Using an image-intensified microscopy system, we observed that our fluorescent TSH bound specifically to diffusely distributed membrane receptors on live rat or bovine embryo thyroid cells grown in culture. At 37 degrees C the fluorescent hormone formed visible patches which were internalized and subsequently degraded. Hence, TSH, like other polypeptide hormones, is internalized by a process of receptor-mediated endocytosis. The addition of bivalent antibodies against the fluorophore rhodamine, to thyroid cells that were previously exposed to sub-optimal concentrations of rhodamine TSH, elicited maximal stimulation of the thyroid adenyl cyclase. Monovalent Fab' fragments did not enhance the response of rhodamine TSH. Therefore we conclude that enhanced surface clustering of TSH molecules increases their capacity to stimulate the adenyl cyclase of thyroid cells.