Ann Janovick J.,ONPRC |
Michael Conn P.,ONPRC
Methods in Enzymology | Year: 2010
The gonadotropin-releasing hormone (GnRH) receptor (GnRHR), because of its small size among G-protein-coupled receptors (GPCRs), is amenable to facile preparation of mutants. This receptor is used in our laboratory as a structural model for this super-family of protein receptors and has helped us understand the requirements for proper trafficking. We have demonstrated that pharmacoperones ("pharmacological chaperones"), small target-specific drugs that diffuse into cells, are capable of rescuing misfolded and misrouted GnRHR mutants and restoring them to function. By rescuing these proteins, these drugs enable the plasma membrane expression of such mutants in living cells and allow examination of mutants that would otherwise be retained in the endoplasmic reticulum and would not be available for ligand binding and signal transduction. As an example of the efficacy of this method, we have shown that mutant E90K, which breaks a salt bridge (E90-K121) normally found in the GnRHR, results in constitutive activity when rescued by pharmacoperones. A second method of rescue, involving a mutation that increases the expression of GnRHRs, is shown to have a similar effect. Normally, in the absence of rescue by either of these methods, this mutant, associated with human hypogonadotropic hypogonadism, is misrouted and this constitutive activity has gone unrecognized. This observation [Janovick, J. A., and Conn, P. M. (2010). Salt bridge integrates GPCR activation with protein trafficking. Proc. Natl. Acad. Sci. USA107, 4454-4458.] showed that the cell normally recognizes this protein as defective and prevents its routing to the plasma membrane. © 2010 Elsevier Inc.
Eghlidi D.H.,ONPRC |
Haley G.E.,ONPRC |
Haley G.E.,Oregon Health And Science University |
Noriega N.C.,University of California at Davis |
And 3 more authors.
Endocrinology | Year: 2010
The neuropeptides kisspeptin, neurokinin B, and dynorphin A (collectively abbreviated as KNDy) are, respectively, encoded by KiSS-1, NKB, and PDYN and are coexpressed by neurons of the hypothalamic arcuate nucleus (ARC). Here, using quantitative real-time PCR, we examined age-related changes in the expression of genes encoding KNDy and associated receptors G protein-coupled receptor 54 (encoded by GPR54), neurokinin 3 receptor (encoded by NK3), and κ-opioid receptor (encoded by KOR), in the female rhesus macaque ARC-median eminence (ARC-ME). Expression of KiSS-1 and NKB was highly elevated in old perimenopausal compared with young or middle-aged premenopausal animals. To test whether these age-related changes could be attributed to perimenopausal loss of sex steroids, we then examined KNDy, GPR54, NK3, and KOR expression changes in response to ovariectomy (OVX) and exposure to 17β-estradiol (E2). Short-term (7 months) OVX (with or without 1 month of estrogen replacement) failed to modulate the expression of any of the KNDy-related genes. In contrast, long-term (∼4 yr) OVX significantly increased KiSS-1 and NKB expression, and this was reversed by E2 administration. Finally, we examined the expression of KNDy-related genes in young adult females during the early follicular, late follicular, or midluteal phases of their menstrual cycle but found no difference. Together, the results suggest that short-term alterations in circulating E2 levels, such as those occurring during the menstrual cycle, may have little effect on the ARC-ME expression of KNDy and associated receptors. Nevertheless, they clearly demonstrate that loss of ovarian steroid negative feedback that occurs during perimenopause plays a major role in modulating the activity of KNDy circuits of the aging primate ARC-ME. Copyright © 2010 by The Endocrine Society.