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Cloning and Expression of the Glucocorticoid Receptor from the Squirrel Monkey (Saimiri boliviensis boliviensis), a Glucocorticoid-Resistant Primate¹

Departments of Pharmacology (P.D.R., J.G.S.), Comparative Medicine (J.G.S.), and Biochemistry and Molecular Biology (S.J.P.), University of South Alabama College of Medicine, Mobile, Alabama 36688

Address all correspondence and requests for reprints to: Jonathan G. Scammell, Ph.D., Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688.

New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent K_d, 20.9 ± 1.8 and 4.3 ± 0.2 nmol/L, respectively; n = 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528–777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser⁵⁵¹ to Thr, Ser⁶¹⁶ to Ala, Ala⁶¹⁸ to Ser, and Ile⁷⁶¹ to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421–486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the human sequence were found in the N-terminal region (amino acids 1–421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe⁷⁷⁴ was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent K_d, 5.9 ± 1.2 and 4.3 ± 0.5 nmol/L, respectively; n = 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent K_d, 20.4 ± 2.0 nmol/L; n = 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function.

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