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Gonadotropin-releasing hormone receptor trafficking may explain the relative resistance to pituitary desensitization in mares
Theriogenology 58 (2002) 523±526
Abstract
Gonadotropin-releasing hormone receptor traf®cking may explain the relative resistance to pituitary desensitization in mares M.B. Porter*, D.C. Sharp Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA
1. Introduction It is recognized that mares are resistant to desensitization of the pituitary in response to continuous GnRH administration relative to other species. Our laboratory has demonstrated that continuous infusion of GnRH (110 mg h 1) for as long as 5 days did not result in extinction of luteinizing hormone (LH) secretion [1]. However, the mechanism for this resistance is not well understood. An understanding of this phenomenon is important in the quest for biological approaches to regulation of reproductive events, such as ovulation induction. To that end, we investigated the structure and intracellular traf®cking of the equine GnRH receptor. 2. Materials and methods Equine pituitaries were obtained at slaughter and either quick frozen for mRNA extraction or placed in sterile transport medium and bisected into 1 mm 1 mm pieces after removal of the neural lobe tissue. 2.1. Receptor structure The equine GnRH receptor was cloned and sequenced by screening an equine pituitary cDNA library constructed and supplied by Dr. Michael Wolfe (Kansas State Medical Center). The library was screened using a portion of the GnRH receptor sequence obtained by reverse transcription±polymerase chain reaction (RT±PCR) in our laboratory.
* Corresponding author. E-mail address: [email protected] (M.B. Porter).
0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 7 7 0 - 7
524
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
2.2. Receptor traf®cking For binding and traf®cking studies, pituitaries in transport media were dispersed with collagenase, viability determined and counted. Pituitary cell surface binding was studied either by incubating whole dispersed cells or by incubating pituitary membrane preparations with radiolabeled D-alanine GnRH. Receptor-mediated endocytosis (internalization) was studied by incubating pituitary cells with radiolabeled D-alanine GnRH at 4 8C until reaching equilibrium, then moving the cells to 37 8C to permit internalization. Cells were returned to 4 8C at various times (0, 20, 40, 60, 120, and 180 min) to stop further internalization, and centrifuged at high speed, brie¯y. Surface-bound radiolabeled Dalanine was removed with ice-cold acetic acid, and internalized label was assessed by centrifugation and counting the pelleted cells. The endocytotic rate constant (ke) was derived by plotting concentration of ligand±receptor complex (intracellular) over surface receptor complex versus time, using non-linear regression techniques (PRISM). The slope of the line was equal to the endocytic rate. Additionally, simultaneous visualization of GnRH and the GnRH receptor in equine pituitary cells was performed with electron microscopy using colloidal gold-labeled antibodies against GnRH and GnRH receptor. Cells were exposed to unlabeled GnRH (10 8 M) at 37 8C for 0, 10, 20, 40, 60, 120, 180, 240, 300, and 420 min. At each time point, cells were brie¯y centrifuged and ®xed in 4% paraformaldehyde and osmium tetroxide for electron microscopy. Fixed cells were exposed to the colloidal gold-labeled antibodies which permitted simultaneous visualization of ligand and receptor having micrometer particle size. 3. Results 3.1. Receptor structure The cloned equine GnRH receptor consisted of 1159 nucleotides encoding a protein of 328 amino acids. The protein exhibits high homology (>85%) with other mammalian GnRH receptor sequences, and exhibited conservation of key amino acids that are believed to be important for membrane receptor binding, transmembrane existence, G-protein association, and phosphorylation. Similar to other mammalian GnRH receptors, but unlike other G-protein associated membrane receptors, the equine GnRH receptor had no intracellular carboxy-terminal tail. Despite the high homology with other mammalian GnRH receptors, however, the equine GnRH receptor exhibited ®ve amino acids (Ser17, Ala26, His61, Asn69, and Phe226) that were different from all other species for which the sequence is known. These amino acids reside within the N-terminus (Ser17 and Ala26), the ®rst intracellular loop (His61 and Asn69), and the ®fth transmembrane domain (Phe226). 3.2. Receptor traf®cking Scatchard plot analysis of initial binding revealed a dissociation constant (KD) of 1:5 10 9 and 5:7 10 9 in whole cells and in cell membrane preparations, respectively. After moving cells to 37 8C and permitting internalization and recycling of receptor, cell
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
525
surface binding decreased slightly between 0 and 20 min, but binding increased thereafter, reaching steady-state at 200% initial binding by 60 min. The internalized radioligand reached steady-state within 30 min and was approximately 30% of total cell-associated ligand. The value of ke was 0.004 min 1 (average of two experiments). With electron microscopy, both GnRH and GnRH receptor were visualized within the cell interior at 10 min after GnRH exposure. GnRH receptor was visualized on the surface at all time points after that, up to 7 h. 4. Discussion The different response to administration of GnRH excessively or continuously in horses compared with other species could re¯ect differences in structure or intracellular traf®cking of the equine GnRH receptor. The fact that equine GnRH receptor gene sequence is highly homologous to known sequences of other animals does not necessarily support that idea. Conservation of amino acids in areas known to be important in ligand binding, signal transduction, and intracellular traf®cking would seem to suggest that an explanation for the tolerance of equine pituitary cells to continuous GnRH administration lies in some other feature of the equine reproductive system. It is of extreme interest to note that ®ve of the amino acids observed in the equine GnRH receptor sequence do not correspond to amino acids in the equivalent location in any other species for which the sequence is known. Whether these few substitutions account for some of the functional differences cannot be determined from the current data. However, it is clear from a variety of published experiments that single amino acid substitutions can lead to dramatic changes or reductions in GnRH receptor function in species tested. Studies of cell surface binding, however, provided at least a partial explanation for ability of equine pituitaries to resist desensitization when exposed to continuous GnRH. These studies indicated that the af®nity of the equine GnRH receptor for its ligand is similar to that reported for other species, and is not unusual in that regard. However, the seminal observation is that cell surface binding did not decline in response to GnRH exposure, as is expected for all other species tested to date. Rather, cell surface binding increased over a 2-h period, indicating that the equine pituitary cell has the ability to provide GnRH receptors to the cell surface in the face of continuous ligand stimulation. Furthermore, derivation of the endocytotic rate indicated that the equine GnRH receptor is internalized at a rate considerably slower than endocytotic rates reported for other species. Several possibilities for resupply of receptors at the surface exist: (1) recycling of existing receptor back to the surface, (2) uncovering of surface receptor previously incapable of binding (occult receptor), or (3) synthesis of new receptor. The time frames studied in this investigation are relatively short, making this third suggested mechanism unlikely. Electron microscopic studies, while non-quantitative, supported the above observations in that GnRH receptor was visualized at the cell surface at all time points studied. We cannot determine from these studies the exact mechanism(s) by which equine pituitary cells maintain a supply of GnRH receptors at the cell surface. Our studies indicated a slow rate of receptor-mediated endocytosis. This observation apparently contributes to the seminal observation that equine pituitary cells maintained the ability
526
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
to bind radiolabeled GnRH in the presence of excess unlabeled GnRH. Whether the few amino acid substitutions that are distinct in the equine GnRH receptor gene sequence provide this capability, or whether there is something inherent in the equine cell itself, remains to be determined. Studies using transfection of the equine GnRH receptor gene sequence into immortalized cell lines are underway to address that question. Finally, the relative resistance to pituitary attenuation by continuous or excessive GnRH by the equine pituitary may re¯ect an evolutionary need for prolonged gonadotropin stimulation to assist in ovarian remodeling for follicular development and ovulation. References [1] Porter MB, Cleaver BD, Peltier M, Robinson G, Thatcher WW, Sharp DC. Comparative study between pony mares and ewes evaluating gonadotrophic response to administration of gonadotrophin-releasing hormone. J Reprod Fertil 1997;110:219±29.
Abstract
Gonadotropin-releasing hormone receptor traf®cking may explain the relative resistance to pituitary desensitization in mares M.B. Porter*, D.C. Sharp Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA
1. Introduction It is recognized that mares are resistant to desensitization of the pituitary in response to continuous GnRH administration relative to other species. Our laboratory has demonstrated that continuous infusion of GnRH (110 mg h 1) for as long as 5 days did not result in extinction of luteinizing hormone (LH) secretion [1]. However, the mechanism for this resistance is not well understood. An understanding of this phenomenon is important in the quest for biological approaches to regulation of reproductive events, such as ovulation induction. To that end, we investigated the structure and intracellular traf®cking of the equine GnRH receptor. 2. Materials and methods Equine pituitaries were obtained at slaughter and either quick frozen for mRNA extraction or placed in sterile transport medium and bisected into 1 mm 1 mm pieces after removal of the neural lobe tissue. 2.1. Receptor structure The equine GnRH receptor was cloned and sequenced by screening an equine pituitary cDNA library constructed and supplied by Dr. Michael Wolfe (Kansas State Medical Center). The library was screened using a portion of the GnRH receptor sequence obtained by reverse transcription±polymerase chain reaction (RT±PCR) in our laboratory.
* Corresponding author. E-mail address: [email protected] (M.B. Porter).
0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 7 7 0 - 7
524
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
2.2. Receptor traf®cking For binding and traf®cking studies, pituitaries in transport media were dispersed with collagenase, viability determined and counted. Pituitary cell surface binding was studied either by incubating whole dispersed cells or by incubating pituitary membrane preparations with radiolabeled D-alanine GnRH. Receptor-mediated endocytosis (internalization) was studied by incubating pituitary cells with radiolabeled D-alanine GnRH at 4 8C until reaching equilibrium, then moving the cells to 37 8C to permit internalization. Cells were returned to 4 8C at various times (0, 20, 40, 60, 120, and 180 min) to stop further internalization, and centrifuged at high speed, brie¯y. Surface-bound radiolabeled Dalanine was removed with ice-cold acetic acid, and internalized label was assessed by centrifugation and counting the pelleted cells. The endocytotic rate constant (ke) was derived by plotting concentration of ligand±receptor complex (intracellular) over surface receptor complex versus time, using non-linear regression techniques (PRISM). The slope of the line was equal to the endocytic rate. Additionally, simultaneous visualization of GnRH and the GnRH receptor in equine pituitary cells was performed with electron microscopy using colloidal gold-labeled antibodies against GnRH and GnRH receptor. Cells were exposed to unlabeled GnRH (10 8 M) at 37 8C for 0, 10, 20, 40, 60, 120, 180, 240, 300, and 420 min. At each time point, cells were brie¯y centrifuged and ®xed in 4% paraformaldehyde and osmium tetroxide for electron microscopy. Fixed cells were exposed to the colloidal gold-labeled antibodies which permitted simultaneous visualization of ligand and receptor having micrometer particle size. 3. Results 3.1. Receptor structure The cloned equine GnRH receptor consisted of 1159 nucleotides encoding a protein of 328 amino acids. The protein exhibits high homology (>85%) with other mammalian GnRH receptor sequences, and exhibited conservation of key amino acids that are believed to be important for membrane receptor binding, transmembrane existence, G-protein association, and phosphorylation. Similar to other mammalian GnRH receptors, but unlike other G-protein associated membrane receptors, the equine GnRH receptor had no intracellular carboxy-terminal tail. Despite the high homology with other mammalian GnRH receptors, however, the equine GnRH receptor exhibited ®ve amino acids (Ser17, Ala26, His61, Asn69, and Phe226) that were different from all other species for which the sequence is known. These amino acids reside within the N-terminus (Ser17 and Ala26), the ®rst intracellular loop (His61 and Asn69), and the ®fth transmembrane domain (Phe226). 3.2. Receptor traf®cking Scatchard plot analysis of initial binding revealed a dissociation constant (KD) of 1:5 10 9 and 5:7 10 9 in whole cells and in cell membrane preparations, respectively. After moving cells to 37 8C and permitting internalization and recycling of receptor, cell
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
525
surface binding decreased slightly between 0 and 20 min, but binding increased thereafter, reaching steady-state at 200% initial binding by 60 min. The internalized radioligand reached steady-state within 30 min and was approximately 30% of total cell-associated ligand. The value of ke was 0.004 min 1 (average of two experiments). With electron microscopy, both GnRH and GnRH receptor were visualized within the cell interior at 10 min after GnRH exposure. GnRH receptor was visualized on the surface at all time points after that, up to 7 h. 4. Discussion The different response to administration of GnRH excessively or continuously in horses compared with other species could re¯ect differences in structure or intracellular traf®cking of the equine GnRH receptor. The fact that equine GnRH receptor gene sequence is highly homologous to known sequences of other animals does not necessarily support that idea. Conservation of amino acids in areas known to be important in ligand binding, signal transduction, and intracellular traf®cking would seem to suggest that an explanation for the tolerance of equine pituitary cells to continuous GnRH administration lies in some other feature of the equine reproductive system. It is of extreme interest to note that ®ve of the amino acids observed in the equine GnRH receptor sequence do not correspond to amino acids in the equivalent location in any other species for which the sequence is known. Whether these few substitutions account for some of the functional differences cannot be determined from the current data. However, it is clear from a variety of published experiments that single amino acid substitutions can lead to dramatic changes or reductions in GnRH receptor function in species tested. Studies of cell surface binding, however, provided at least a partial explanation for ability of equine pituitaries to resist desensitization when exposed to continuous GnRH. These studies indicated that the af®nity of the equine GnRH receptor for its ligand is similar to that reported for other species, and is not unusual in that regard. However, the seminal observation is that cell surface binding did not decline in response to GnRH exposure, as is expected for all other species tested to date. Rather, cell surface binding increased over a 2-h period, indicating that the equine pituitary cell has the ability to provide GnRH receptors to the cell surface in the face of continuous ligand stimulation. Furthermore, derivation of the endocytotic rate indicated that the equine GnRH receptor is internalized at a rate considerably slower than endocytotic rates reported for other species. Several possibilities for resupply of receptors at the surface exist: (1) recycling of existing receptor back to the surface, (2) uncovering of surface receptor previously incapable of binding (occult receptor), or (3) synthesis of new receptor. The time frames studied in this investigation are relatively short, making this third suggested mechanism unlikely. Electron microscopic studies, while non-quantitative, supported the above observations in that GnRH receptor was visualized at the cell surface at all time points studied. We cannot determine from these studies the exact mechanism(s) by which equine pituitary cells maintain a supply of GnRH receptors at the cell surface. Our studies indicated a slow rate of receptor-mediated endocytosis. This observation apparently contributes to the seminal observation that equine pituitary cells maintained the ability
526
M.B. Porter, D.C. Sharp / Theriogenology 58 (2002) 523±526
to bind radiolabeled GnRH in the presence of excess unlabeled GnRH. Whether the few amino acid substitutions that are distinct in the equine GnRH receptor gene sequence provide this capability, or whether there is something inherent in the equine cell itself, remains to be determined. Studies using transfection of the equine GnRH receptor gene sequence into immortalized cell lines are underway to address that question. Finally, the relative resistance to pituitary attenuation by continuous or excessive GnRH by the equine pituitary may re¯ect an evolutionary need for prolonged gonadotropin stimulation to assist in ovarian remodeling for follicular development and ovulation. References [1] Porter MB, Cleaver BD, Peltier M, Robinson G, Thatcher WW, Sharp DC. Comparative study between pony mares and ewes evaluating gonadotrophic response to administration of gonadotrophin-releasing hormone. J Reprod Fertil 1997;110:219±29.