Differential Roles of Exoloop 1 of the Human Follicle-stimulating Hormone Receptor in Hormone Binding and Receptor Activation

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Luteinizing hormone receptor, a G protein-coupled receptor, consists of two halves, the N-terminal extracellular hormone binding domain (exodomain) and the Cterminal membrane-associated, signal-generating domain (endodomain). The exodomain has seven to nine Leu-rich repeats, which are generally thought to form a 1/3 donut-like structure and interact with human choriogonadotropin (hCG). The resulting hCG-exodomain complex adjusts the structure and its association with the endodomain, which results in signal generation in the endodomain. It is unclear whether the rigid 1/3 donut structure could provide the agility and versatility of this dynamic action. In addition, there is no clue as to where the endodomain contact point (the signal modulator) in the exodomain is. To address these issues, the exodomain was examined by Ala scan and multiple substitutions, while receptor peptides were used for photoaffinity labeling and affinity cross-linking. Our results show that the C-flanking sequence (hinge region), Thr 250 -Gln 268 , of the Leu-rich repeats (LRRs) specifically interacts with hCG, preferentially hCG␣. This interaction is inhibited by exoloop 2 of the endodomain but not by exoloops 1 and 3, suggesting an intimate relationship between Thr 250 -Gln 268 , exoloop 2, and hCG. Taken together, our observations in this article suggest a new paradigm that the LRRs contact the front of hCG, while both flanking regions of the LRRs interact with the sides of hCG. This would trap hCG in the 1/3 donut structure of the LRRs and enhance the binding affinity. In addition, mutations of conserved Ser 255 in the sequence can constitutively activate the receptor. This provides a clue for the signal modulator in the exodomain. In contrast, a phenyl or phenolic group is necessary at conserved Tyr 253 for targeting the receptor to the surface.

Section: Resultsmentioning

confidence: 99%

The Role of the Hinge Region of the Luteinizing Hormone Receptor in Hormone Interaction and Signal Generation

Zeng¹,

Phang²,

Song³

et al. 2001

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“…Mutagenesis and Functional Expression of Receptors-Each mutant human LHR and FSHR cDNA was prepared in a pSELECT vector using the non-polymerase chain reaction-based Altered Sites Mutagenesis System (Promega), sequenced, and subcloned into pcDNA3 (Invitrogen) as described previously (40). After subcloning pcDNA3, the mutant cDNAs were sequenced again.…”

Section: Methodsmentioning

confidence: 99%

Hormone Interactions to Leu-rich Repeats in the Gonadotropin Receptors

Song¹,

Ji²,

Beauchamp³

et al. 2001

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The luteinizing hormone receptor (LHR) and folliclestimulating hormone receptor (FSHR) have an ϳ350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the ␤-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these ␤ strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the ␤-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that ␤-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.

“…Each mutant human FSHR cDNA was prepared in a pSELECT vector using the nonPCR-based Altered Sites mutagenesis system (Promega), sequenced on a Beckman CEQ 2000XL capillary sequencer, and subcloned into pcDNA3 (Invitrogen) as described (22). After subcloning pcDNA3, the mutant cDNAs were sequenced again.…”

Section: Mutagenesis and Functional Expression Of Human Fsh Receptor-mentioning

confidence: 99%

Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor

Sohn

Ryu

Sievert

et al. 2002

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The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ϳ330 amino acids, the N-terminal extracellular exodomain and membraneassociated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/ exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of 580 KVPLITVSKAK 590 was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH ␣ but not FSH ␤. This is in contrast to photoaffinity labeling of FSH ␤ by the peptide mimic of the N-terminal region of the receptor. The FSH1 receptor (FSHR) 1 and other glycoprotein hormone (luteinizing hormone/chorionic gonadotropin and thyroid-stimulating hormone) receptors belong to a structurally unique subfamily of G protein-coupled receptors. Unlike other receptor subfamilies, they comprise two equal halves, an N-terminal extracellular half (exodomain) and a C-terminal membraneassociated half (endodomain) (1-4). The exodomain is ϳ350 amino acids long and alone is capable of high affinity hormone binding (5-8) with hormone selectivity (9 -11) but without hormone action (7,12). Receptor activation occurs in the endodomain (13), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (14). Glycoprotein hormones initially bind to the exodomain, and then the resulting hormone/exodomain complex modulates the endodomain (13), which activates adenylyl cyclase (AC) to generate cAMP and phospholipase C␤ (PLC␤) to produce inositol phosphate and diacylglycerol. Therefore, the ternary interactions among the hormone, exodomain, and endodomain are crucial for successful signal generation. However, there is little information on the subject, particularly concerning the FSH receptor. Since the exodomain lacking the endodomain is capable of high affinity hormone binding (5-8, 15), the high affinity hormone binding appears to be independent of the endodomain. Contrary to this view, it has been reported that FSH and human chorionic gonadotropin binding to their cognate receptors is regulated by certain residues of exoloops 2 and 3 of the endodomain (15, 16). Furthermore, the hinge region of the exodomain interacts with exoloop 2 and modulates cAMP induction (17-19). These results suggest that the exodomain interacts with the exoloops and modulates them for signal generation. Yet it is unclear whether the hormone complexed with the exodomain also contacts the exoloops.In this study, we set out to investigate whether exoloops interact with the hormone at all. In a step toward this goal, we examined exoloop 3 of FSHR for its involvement in the activation of the two eff...