CLONING AND EXPRESSION OF THE GLUCOCORTICOID RECEPTOR FROM THE SQUIRREL-MONKEY (SAIMIRI-BOLIVIENSIS-BOLIVIENSIS), A GLUCOCORTICOID-RESISTANT PRIMATE

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 lym phocytes is 6-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 an d have compared the sequence to receptor sequences obtained from owl m onkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. Th e squirrel monkey glucocorticoid receptor is approximately 97% identic al in nucleotide and amino acid sequence to the human receptor. The li gand-binding domain (amino acids 528-777) of the squirrel monkey gluco corticoid receptor contains four amino acid differences (Ser(551) to T hr, Ser(616) to Ala, Ala(618) to Ser, and Ile(761) to Leu), all of whi ch are present in owl monkey and cotton-top tamarin receptors. The DNA -binding domain (amino acids 421-486) is completely conserved among hu man, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Tw enty-two differences from the human sequence were found in the N-termi nal region (amino acids 1-421) of the squirrel monkey receptor. None o f the substitutions in the ligand-binding domain matched mutations kno wn to influence binding affinity in other species. To determine whethe r the substitutions per se were responsible for decreased affinity, sq uirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squi rrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe(774) was mutated to Leu (F774L) were similar. When expresse d in the TNT System, squirrel monkey and human glucocorticoid receptor s 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 glucocorticold receptor cannot a ccount for the decreased binding affinity of these receptors in squirr el monkey cells. Rather, the binding affinity is probably influenced b y the expression of cytosolic factors that affect glucocorticoid recep tor function.