Membrane organization of the gonadotropin releasing hormone receptor

Abstract

Sometimes referred to as the "first hormone of reproduction", GnRH is the key hypothalamic input that stimulates and maintains the functional integrity of gonadotrope cells in the anterior pituitary gland. Thus, the interaction of GnRH with its cognate pituitary receptor is central to the normal reproductive function of mammals. Accordingly, enormous effort has been expended toward understanding physiological, cellular and molecular regulation of GnRH and the GnRH receptor (GnRHR). We have found that the GnRHR and down stream signaling intermediates, including c-raf kinase are constitutive residents of discrete membrane domains termed lipid rafts. These raft domains are thought to consist of tightly packed sphingolipid and cholesterol. From a signaling standpoint, lipid rafts have generated much interest as membrane scaffolds for spatial and temporal organization of cell-surface receptors and their downstream effectors. Towards this end, we have found that disruption of lipid rafts by cholesterol depletion in αT3-1 cells using methyl-β-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. We were able to rescue raft localization and GnRHR signaling to ERK upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low-density membrane microdomains appears to be critical for the ability of GnRH to propagate an intracellular signal to the level of MAP kinase activation. Given the central role lipid rafts play in the activation of the GnRHR, another key issue is the identity of structural motifs that direct lipid raft localization of the GnRHR. Because the absence of an intracellular carboxyl terminal domain is one of the more conspicuous and unique features of the GnRHR, we were intrigued with the possibility that the association of the GnRHR with lipid rafts may reflect both a loss (C-terminus) and gain (raft association address) of structural characteristics. To address this, we fused either the full length C-terminus from the non-raft associated LH Receptor (LHR) (GnRHR-LF) or a truncated (t631) LHR C-terminus, (GnRHR-LT) to the GnRHR. These chimeric receptors are trafficked to the plasma membrane, bind ligand and display increased agonist induced receptor internalization but do not partition into lipid rafts. Thus, a heterologous C-terminus from a non-raft associated GPCR redirects localization of the GnRHR to non-raft domains. In contrast to the murine GnRHR, the catfish GnRHR (cfGnRHR) possesses an intracellular C-terminus. We find that the cfGnRHR is localized to lipid rafts and that the cfGnRHR C-terminus does not alter raft localization of the mammalian receptor. Consistent with placement in different lipid microenvironments within the plasma membrane, fluorescence recovery after photobleaching (FRAP) reveals different lateral diffusion phenotypes of the raft associated GnRHR and cfGnRHR vs. the non-raft associated GnRHR-LF fusion protein. We conclude that while an intracellular C-terminus is capable of redirecting the GnRHR to non-raft compartments, this is not a generalized feature of GPCR C-terminal tails. In summary, while the research presented in this dissertation confirms the constitutive presence of GnRHR to lipid rafts, the localization is not simply due to the loss of an intracellular C-terminus.