Studies on the desensitization of the cyclic AMP response to thyrotropin in thyroid tissue

B. Rapoport*, S. Filetti, N. Takai and P. Seto Medical Service (l l lF), Veterans'Administration Hospital, 4150 Clement Street, San Francisco, CA 94121, USA Received 9 July 1982 Thyroid cell Thyrotropin Desensitization A denylate cyclase N-protein A DP-ribosylation 1. INTRODUCTION 2. MATERIALS AND METHODS Prior exposure of thyroid tissue to thyrotropin (TSH) in vitro leads to desensitization, of the aden- ylate cyclase response to TSH stimulation [1-6]. Recently, it has been suggested that ADP-ribosyla- tion reactions may be involved in the TSH desensi- tization process [7,8]. Circumstantial support for this thesis was provided by the finding that TSH enhances ADP-ribosylation of acceptor proteins in isolated thyroid cells [9]. However, the nature of these proteins has not been established. One pro- tein of particular interest is the adenylate cyclase regulatory subunit (N, or G/F, protein), which is ADP-ribosylated during cholera toxin activation of adenylate cyclase (reviewed [10,11]. In [8] it was proposed that N protein ADP-ribosylation is im- portant in TSH desensitization in the thyroid. However, epinephrine or prostaglandin El desen- sitization in $49 mouse lymphoma cells is not asso- ciated with altered N protein function [12]. These studies were undertaken to examine, in the thyroid, whether N protein is specifically ADP- ribosylated by TSH. In addition, the functional ac- tivity of the N protein, as well as of the adenylate cyclase catalytic unit (C) was tested in membranes from thyroid cells desensitized to TSH. The data obtained suggest that N protein ADP-ribosylation is not responsible for TSH desensitization, and help localize the 'defect' in TSH desensitization to TSH receptor (R) function, or to R-N interaction. *To whom reprint requests should be addressed 2.1. Cholera toxin-induced ADP-ribosylation of pu- rified thyroid plasma membranes Dog thyroid plasma membranes were prepared by discontinuous sucrose gradient sedimentation 113,14]. The membranes (0.6 mg protein) were then treated for 30 min at 30°C with pre-activated cholera toxin (10 /~g/ml) in 100 /~M GTP and 10/~M [32p]NAD+, in 200/zl final vol. [14]. Sam- ples (10-20 /4) were subjected to SDS-poly- acrylamide slab gel electrophoresis (7.5-15% gra- dient) [16]. Mr standards (10 000-70 000 Mr) were from Sigma Chemical Co. (catalogue no. MW- SDS-70). The gels were stained, dried and exposed to Kodak X-Omat XAR-5 film. 2.2. TSH-induced ADP-ribosylation of dog thyroid cells The method in [9] was employed. The cells were diluted in ice-cold HME buffer (20 mM Hepes, 2 mM MgC12, 1 mM EDTA, pH 7.4), then cen- trifuged for 10 rain at 1000 x g. The pellet was re- suspended in 3 ml HME buffer, supplemented with 1 mM ADP-ribose, 2 mM 3-isobutyl-l-meth- ylxanthine, 0.1 mM phenylmethylsulfonylfluoride. After homogenization with a Polytron the material was centrifuged (1000 x g for 15 min). The pellet was rehomogenized and re-centrifuged as above. The combined supernatants were centrifuged (30 000 x g for 30 min) and the pellet was sol- ubilized in a minimum volume (100-200 /~1) of SDS-containing buffer for slab gel electrophoresis [16]. Published by Elsevier Biomedical Press 00145793/82/0000--0000/$2.75 ® 1982 Federation of European Biochemical Societies 23