Abstract
This report explores the biochemical basis for clonal variation in adrenocorticotropin (ACTH)-sensitive adenylate cyclase activity in the Y1 mouse adrenocortical tumor cell line. We demonstrate that the level of a specific protein, designated p68, is significantly correlated with the ability of adrenocorticotropin to stimulate adenylate cyclase activity among Y1 subclones (p = 0.004; r = 0.65). p68 was characterized by its molecular weight in sodium dodecyl sulfate polyacrylamide gels (Mr = 68,000) and by its isoelectric point as determined by two-dimensional gel electrophoresis (pI = 7.2). On two-dimensional gels, the protein migrated as a major spot with satellite spots 0.1 pH unit on either side. Homogenates and plasma membrane fractions from clones highly responsive to ACTH had large amounts of p68. In homogenates from highly responsive clones p68 represented 10 to 12% of the total protein. Homogenates and plasma membrane fractions from clones insensitive to ACTH were deficient in p68. In homogenates from the insensitive clones Y6 and OS3, p68 represented less than or equal 0.8% of the total protein. A somatic cell hybrid, formed by fusion of these two ACTH-insensitive clones recovered ACTH-sensitive adenylate cyclase activity and concomitantly expressed appreciable levels of p68. It is suggested that p68 may regulate the transfer of information from the occupied ACTH receptor ot the catalytic subunit of adenylate cyclase.
Highlights
= 0.004; r = 0.65). p68 was characterized byits molec- (Rae et al, 1979b; Schimmer, 1972), suggestingthat the adeular weight in sodium dodecyl sulfate polyacrylamide nylate cyclase system was partially intact
We have found a significant correlation between the ability of ACTH to stimulate adenylate cyclase activity and the amount of a specific protein, p68, among subclones of the Y1 adrenocortical tumor cell line
The hybrid clone 2 remained insensitive to ACTH and retained the low levels ofp68 associated with unresponsive clones (Table 11).The mechanism responsible for the reconstitution of ACTH-sensitive adenylate cyclase activity in the hybrid clone 1 is unknown; preliminary evidence suggests that restoration of ACTH sensitivity through cell fusion isan infrequent event and, probably not due to asimple complementation of two different mutations
Summary
ACTH-responsive subclones were isolated from the Y1-ATCC line by single cell-plating techniques and include: Yl-BS1 (Schimmer, 1979), Y1-BS8, and Y1-BS11. Other clones used in this study were two spontaneous, ACTH-insensitivevariants, Y6 and OS3 (Schimmer, 1969, 1972);two cell limes deficient in hypoxanthine-guanosine phosphoribosyltransferase activity, Y1-HGPRT- and OS3-HGPRT-. The abbreviations used are: ACTH, adrenocorticotropin; methods of cell culturehave been detailed elsewhere (Schimmer, GPP(NH)P, guanyl-5”yl imidodiphosphate; SDS, sodium dodecyl 1979). Adenylate cyclase activity in ACTH-responsive and -insensitive YI adrenal clones. Adenylate cyclase activity was measured in the presence of 10p~ GPP(NH)P; ACTHI-,,(20 p ~ a)nd NaF (15 mM) were added as indicated. Values shown are averages of duplicate or triplicate samples from representative experiments and are expressed as picomoles of cAMP formed per rnin per mg of protein
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