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

Abstract

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 Kd, 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 Kd, 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 Kd, 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.

Ser/Thr Protein Phosphatase Type 5 (PP5) Is a Negative Regulator of Glucocorticoid Receptor-Mediated Growth Arrest

“…Receptor Binding Studies, Transfections, and Luciferase Assays. Intact cell binding studies were conducted with [ 3 H]dexamethasone as described previously (41). Transfections and luciferase assays were conducted as described by Jones et al (42), using a mouse mammary tumor virus-luciferase reporter plasmid (MMTV-Luc).…”

Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement

“…Squirrel monkeys have very high levels of circulating corticosteroid and require much higher levels of hormone for GR-dependent transactivation. However, the cloned Bolivian squirrel monkey GR expressed in vitro was found to bind dexamethasone with the same affinity as the human GR [39]. Interest then focused on the high ratio of FKBP51 to FKBP52 found in squirrel monkey cells [40 -42].…”

Phosphorylation and Inhibition of Rat Glucocorticoid Receptor Transcriptional Activation by Glycogen Synthase Kinase-3 (GSK-3)

“…Sequence comparison between GRs isolated from different species shows that the primary amino acid sequence surrounding and including rat GR Thr 171 (residues 164 through 173) is conserved among rodents, including rat, mouse, and guinea pig (40,41,43). The equivalent region from human, squirrel monkey, owl monkey, and cotton-top tamarin GR remains conserved among primates, but has diverged from rodents (42,48,49). Why this region of GR has diverged between primates and rodents, while the other major phosphorylation sites (Ser 224 , Ser 232 , and Ser 246 ) are conserved remains unclear, but likely reflects alternative strategies adopted by each species to regulate GR action.…”