HETEROGENEITY OF INSULIN-RECEPTORS IN RAT-TISSUES AS DETECTED WITH THE PARTIAL AGONIST B29,B29'-SUBEROYL-INSULIN

Using the insulin receptor partial agonist B29,B29'-suberoyl-insulin, a covalently dimerized insulin derivative. we previously demonstrated a heterogeneity of signal transduction by insulin receptors in two cel l systems. The present study was designed to characterize the heteroge neity of insulin receptors in different rat tissues with this agent. B inding of I-125-insulin to insulin receptors and its inhibition by B29 ,B29'-suberoyl-insulin or by unlabeled insulin were assayed in plasma membranes from brain. spleen, adipocytes, and liver. IC50 values of B2 9,B29'-suberoyl-insulin were different in all tissues investigated (br ain < spleen < adipocytes < liver). In contrast, IC50 values of insuli n were identical, with the exception of spleen membranes (spleen < bra in = adipocytes = liver). Furthermore, the IC50 ratios (B29 dimer/insu lin) were significantly different, ranging from 0.7 (brain) to 12.8 (l iver). Solubilization and partial purification of insulin receptors fa iled to abolish the marked difference between brain and liver (IC50 ra tios of 1.8 and 7.1, respectively). The apparent molecular masses of t he a subunits of insulin receptors, as labeled with a photoreactive in sulin derivative, appeared identical in liver and spleen but were sign ificantly lower in adipocytes and brain (liver = spleen > adipocytes > brain). The tissue-specific expression of the known insulin receptor isoforms generated by alternative splicing (insulin receptor types A a nd B), as assessed by polymerase chain reaction amplification with oli gonucleotide primers flanking exon II, was not correlated with the dif ferences in the IC50 values and ratios for insulin and B29,B29'-subero yl-insulin. Furthermore, IC50 values of both insulin and the B29 dimer were 3-fold lower in membranes from Rat1 cells overexpressing insulin receptor type A, compared with membranes with insulin receptor type B ; the IC50 ratios were identical. No additional alternative splicing o f insulin receptor mRNA was found by polymerase chain reaction amplifi cation and digestion with HaeIII and AluI of seven overlapping domains of the receptor alpha subunit. These data suggest a heterogeneity of insulin receptors in rat tissues that is unrelated to alternative spli cing of the insulin receptor gene.