Two mutations of the lutropin/choriogonadotropin receptor that impair signal transduction also interfere with receptor-mediated endocytosis.

Previous results from this laboratory have shown that human kidney (293) cells transfected with the rat follitropin receptor (rFSHR) internalize agonist (i.e. human follitropin, hFSH) at a rate similar to that of other agonist-G protein-coupled receptor complexes while 293 cells transfected with the rat lutropin/choriogonadotropin receptor (rLHR) internalize agonist (human choriogonadotropin, hCG) at a rate that is about 1 order of magnitude slower. Taking advantage of this difference and the high degree of homology between the rLHR and rFSHR, we have now used chimeras of these two receptors to begin to delineate structural features that influence their internalization. Analysis of six chimeras that exchanged only the transmembrane domains (designated FLF and LFL), only the COOH-terminal domains (FFL or LLF) or both domains (FLL or LFF) show that the origin of the extracellular domain is at least as important, if not more, than the origin of the transmembrane and COOH-terminal domains in determining the rate of internalizationof the gonadotropin receptors. Thus, the rates of internalization of agonist internalization mediated by FFL, FLF, and FLL more closely resemble rFSHR than rLHR, while the rates of agonist internalization mediated by LLF, LFL, and LFF more closely resemble rLHR than rFSHR.The importance of the extracellular domain was also evident even upon overexpression of arrestin-3, a protein that enhances the rate of internalization of the wild-type receptors and chimeras by binding to their intracellular regions.

Section: Discussionsupporting

confidence: 86%

Section: Construction and Signaling Properties Of Gonadotropinmentioning

confidence: 88%

See 1 more Smart Citation

The Rate of Internalization of the Gonadotropin Receptors Is Greatly Affected by the Origin of the Extracellular Domain

Nakamura

Liu

Ascoli

1999

“…Thus, the rate of internalization of the free LHR is slower than the rate of internalization of the agonist-receptor complex (8,10) and the rate of internalization of a complex formed by the LHR and a weak partial agonist is slower than that of the complex formed by the LHR and a full agonist (11). Likewise, mutations of the LHR that impair signal transduction or induce constitutive activation impair or enhance agonist-induced internalization, respectively (10,12). On the other hand, mutations of the LHR that do not affect agonist-induced activation, but impair agonist-induced rLHR phosphorylation, also impair agonist-induced internalization (13,14).…”

Section: Agonist Binding To G Protein-coupled Receptors (Gpcr)mentioning

confidence: 99%

Seven Non-contiguous Intracellular Residues of the Lutropin/Choriogonadotropin Receptor Dictate the Rate of Agonist-induced Internalization and Its Sensitivity to Non-visual Arrestins

Nakamura¹,

Liu

Ascoli

2000

The amino acid sequences of the human (h) and rat (r) lutropin/choriogonadotropin receptors (LHR) are 87% identical, but the rate of agonist-induced internalization of the hLHR is ϳ7 times faster than that of the rLHR. Chimeras of the hLHR and the rLHR showed that this rate is dictated by the serpentine domain and the cytoplasmic tail. Further mutational analysis identified seven residues, two adjacent residues in the second intracellular loop (Val/Gln in the rLHR and Ile/His in the hLHR), four non-contiguous residues in the third intracellular loop (Arg/Gln/Thr/Pro in the rLHR and Lys/Arg/ Met/Thr in the hLHR), and one in the C-terminal tail (Leu in the rLHR and Phe in the hLHR), that are necessary and sufficient to impart the slow rate of internalization of the rLHR and the fast rate of internalization of the hLHR. The internalization of the rLHR and the hLHR display different sensitivities to the non-visual arrestins. Therefore, we also tested if the simultaneous exchange of these seven residues resulted in the exchange of this property. Since this was found to be the case, we propose that these seven residues identified here form a non-visual arrestin-binding site.Agonist binding to G protein-coupled receptors (GPCR) 1 is quickly followed by the internalization of the receptor or the agonist-receptor complex. This process is facilitated by the GPCR phosphorylation that follows agonist-induced activation, and it usually requires the formation of a binary complex between the agonist-activated GPCR and the non-visual arrestins. The non-visual arrestins bind clathrin with high affinity and serve as adaptor molecules that target agonist-activated phosphorylated GPCRs to clathrin-coated pits (1-4).The LHR is a member of the rhodopsin-like subfamily of GPCRs (5), and the agonist-LHR complex is internalized (6 -8) via clathrin-coated pits (9) following agonist-induced activation. Like other GPCRs, the agonist-induced LHR activation and phosphorylation, as well as the interaction of the LHR with a non-visual arrestin, can now be recognized as important steps in agonist-induced internalization of the LHR. Thus, the rate of internalization of the free LHR is slower than the rate of internalization of the agonist-receptor complex (8, 10) and the rate of internalization of a complex formed by the LHR and a weak partial agonist is slower than that of the complex formed by the LHR and a full agonist (11). Likewise, mutations of the LHR that impair signal transduction or induce constitutive activation impair or enhance agonist-induced internalization, respectively (10, 12). On the other hand, mutations of the LHR that do not affect agonist-induced activation, but impair agonist-induced rLHR phosphorylation, also impair agonist-induced internalization (13,14). Finally, the internalization of the agonist-LHR complex can be inhibited by co-transfection with dominant-negative mutants of the non-visual arrestins or by co-transfection with a dominant-negative mutant of dynamin (14, 15). Whereas most internalized GPCRs recycle qui...

“…Another notable difference between the LHR and other GPCRs is on the rate of internalization. The t1 ⁄2 of internalization of most GPCRs that are internalized by a nonvisual arrestin/clathrin/dynamin-dependent pathway is 10 -15 min (11,12), while the t1 ⁄2 of internalization of the LHR, which is also internalized by a nonvisual arrestin/clathrin/ dynamin-dependent pathway (2, 3) is 20 -60 min in target cells (5,8,13) and 100 -140 min in 293 cells expressing the recombinant rLHR (3,14,15). It is not known if this slower rate of internalization of the rLHR-agonist complex is a reflection of a weak interaction of the rLHR with the nonvisual arrestins or if it is due to the involvement of other adapter proteins (see Ref.…”

Section: The Lutropin/choriogonadotropin Receptor (Lhr)mentioning

confidence: 99%

Mutations That Induce Constitutive Activation and Mutations That Impair Signal Transduction Modulate the Basal and/or Agonist-stimulated Internalization of the Lutropin/Choriogonadotropin Receptor

Min¹,

Liu²,

Fabritz³

et al. 1998

Previous results from this laboratory suggested that the same active conformation of the lutropin/choriogonadotropin receptor (LHR) is involved in the stimulation of G proteins and in triggering the internalization of the bound agonist.We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation does not affect the internalization of the free receptor, but it enhances the internalization of the agonist-occupied receptors ϳ3-fold. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ϳ2-and ϳ17-fold, respectively. We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization.Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R (a system where internalization does not occur) respond to hCG with an increase in adenylyl cyclase activity supports this suggestion. The lutropin/choriogonadotropin receptor (LHR)1 is a member of the rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) (reviewed in Ref. 1) that has been shown to mediate the internalization of its two naturally occurring agonists, lutropin and choriogonadotropin (CG). These studies, which have been conducted in target or transfected cells, have shown that the free LHR is randomly distributed in the plasma membrane, but that it clusters in clathrin-coated pits upon agonist activation (2). The clustered agonist-receptor complex is internalized by a dynamin-dependent pathway and traverses the endosomal compartment without agonist dissociation (3-5). Dissociation of the agonist-receptor complex occurs in the lysosomes, where both the agonist and the receptor are degraded (2, 4 -6). The receptor-mediated endocytosis of lutropin and CG serves two important functions in regulating cellular responsiveness. First, the internalization of the receptor-bound agonist effectively stops activation of downstream effectors such as adenylyl cyclase (7). Second, the accumulation of the agonistreceptor complex in the lysosomes promotes receptor degradation and is ultimately responsible for the agonist-induced down-regulation of the cell surface LHR, an outcome that prevents further activation of the cells (4,8,9).The lysosomal accumulation of the rLHR-agonist complex and the involvement of rLHR internalization in the termination of hormone action (i.e. desensitizat...