Lack of a C-terminal Tail in the Mammalian Gonadotropin-releasing Hormone Receptor Confers Resistance to Agonist-dependent Phosphorylation and Rapid Desensitization

Self Cite

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo-and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either ␤-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only ␤-arrestin 2 could enhance TRHR2 internalization. The preference for ␤-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a ␤-arrestin 2 knockout or a ␤-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both ␤-arrestins. The differential ␤-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and ␤-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with ␤-arrestins, could also enhance TRHR2/␤-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the ␤-arrestins and also provides the first evidence that the interactions of TRHRs with ␤-arrestin may be altered by hetero-oligomer formation. G-protein-coupled receptor (GPCR)1 function is underpinned by the formation of protein-protein interactions and complexes that are dynamically regulated by a variety of stimuli. These molecular interactions are involved in receptor recognition, activation, and desensitization and are targets for the majority of current therapeutic compounds utilized to treat a variety of disorders. More recently, the escalating body of evidence for GPCR oligomerization also adds another level of complexity in understanding how GPCRs are activated and signal and traffick in the cell. GPCR hetero-oligomerization may explain the diverse physiological responses obtained from a single ligand. Indeed, a number of GPCR subtypes such as the somatostatin, opioid, angiotensin, and ␤-adrenergic receptors have been shown to form hetero-oligomers that result in a receptor unit with either altered ligand specificity, signaling, or internalization rates (1-5). GPCR activation is also regulated by receptorprotein interactions involved in desensitization and internalization. For the majority of GPCRs, the mechanism of internalization is via the ␤-arrestin and dynamin-dependent pathway where agonist-activated phosphorylated receptors interact with the ␤-arres...

Section: Discussioncontrasting

confidence: 53%

Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

Hanyaloglu¹,

Seeber²,

Kohout³

et al. 2002

Self Cite

“…Moreover, internalization of catfish GnRH-R is ␤-arrestin-dependent (24), whereas internalization of the human GnRH-R is not (25). Thus non-mammalian GnRH-Rs appear to follow the scheme outlined above for ␤-arrestin-mediated desensitization and internalization (and possible ␤-arrestin-mediated signaling), but this is not the case for the mammalian GnRH-Rs investigated to date (20,22).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, all cloned non-mammalian GnRH-Rs possess such tails and, since the serine and threonine residues that are phosphorylated by GRKs are often found within the C-terminal tail, comparative studies have addressed the functional relevance of these structures. These have revealed that mammalian GnRH-Rs do not show rapid homologous desensitization (21)(22), whereas non-mammalian GnRH-Rs do rapidly desensitize (23) and that, although mammalian GnRH-Rs undergo agonist-induced internalization via CCVs, they are internalized much more slowly than nonmammalian GnRH-Rs. Moreover, where investigated, nonmammalian GnRH-Rs show agonist-induced phosphorylation and ␤-arrestin translocation and undergo ␤-arrestin-dependent internalization (24), whereas mammalian GnRH-Rs do not (25).…”

mentioning

confidence: 99%

Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors

Hislop¹,

Everest²,

Flynn³

et al. 2001

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of ␤-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF-and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.

“…In some GPCRs, these phosphorylation events allow for interaction with ␤-arrestins and subsequent receptor deactivation and internalization (25,26). Consistent with the lack of an extensive C-terminal tail, the GnRHR does not appear to undergo arrestindependent internalization or desensitization (27)(28)(29)(30)(31). In fact, the GnRHR has been considered as a naturally occurring internalization resistant mutant (28).…”

mentioning

confidence: 88%

Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK

Navratil

Bliss

Berghorn

et al. 2003

134

105

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of ␣T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of ␣T3-1 cells using methyl-␤-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. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.