The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to G(s)- and G(q/11)-mediated signal transduction pathways: Evidence from loop fragment transfection in GGH3 cells

Alfredo Ulloa-Aguirre, Dinesh Stanislaus, Vivek Arora, Jeffrey Väänänen, Shaun P Brothers, Jo Ann Janovick, P. Michael Conn

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane- proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH31' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both G(s) and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other G(s)-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH31' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH31' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the α(1B)-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach- muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the G(s) protein). Transfection of loop 3i from the D(1A) -dopamine receptor (coupled to the G(s) protein) produced a selective attenuation (40%) in G(s)-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and α(2A)-adrenergic receptor's). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH31' cell line, this loop is also involved in signal transduction mediated through the G(s) protein pathway.

Original languageEnglish
Pages (from-to)2472-2478
Number of pages7
JournalEndocrinology
Volume139
Issue number5
StatePublished - Nov 20 1998
Externally publishedYes

Fingerprint

LHRH Receptors
Transfection
Signal Transduction
GTP-Binding Proteins
Gonadotropin-Releasing Hormone
Inositol Phosphates
Plasmids
Complementary DNA
G-Protein-Coupled Receptors
Proteins
Adrenergic Agents
Cholinergic Agents
Gi-Go GTP-Binding Protein alpha Subunits
Buserelin
Cell Line
Rhodopsin
Dopamine Receptors
Muscarinic Receptors
Adrenergic Receptors
Cell Membrane

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to G(s)- and G(q/11)-mediated signal transduction pathways : Evidence from loop fragment transfection in GGH3 cells. / Ulloa-Aguirre, Alfredo; Stanislaus, Dinesh; Arora, Vivek; Väänänen, Jeffrey; Brothers, Shaun P; Janovick, Jo Ann; Conn, P. Michael.

In: Endocrinology, Vol. 139, No. 5, 20.11.1998, p. 2472-2478.

Research output: Contribution to journalArticle

Ulloa-Aguirre, Alfredo ; Stanislaus, Dinesh ; Arora, Vivek ; Väänänen, Jeffrey ; Brothers, Shaun P ; Janovick, Jo Ann ; Conn, P. Michael. / The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to G(s)- and G(q/11)-mediated signal transduction pathways : Evidence from loop fragment transfection in GGH3 cells. In: Endocrinology. 1998 ; Vol. 139, No. 5. pp. 2472-2478.
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abstract = "The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane- proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH31' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both G(s) and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other G(s)-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH31' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45{\%}) maximally inhibited buserelin-stimulated IP turnover by 20{\%} as well as cAMP accumulation and PRL secretion by 30{\%}. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH31' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the α(1B)-adrenergic (G(q/11)-coupled) receptors resulted in 25-55{\%} inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach- muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40{\%} (both effects mediated through activation of the G(s) protein). Transfection of loop 3i from the D(1A) -dopamine receptor (coupled to the G(s) protein) produced a selective attenuation (40{\%}) in G(s)-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and α(2A)-adrenergic receptor's). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH31' cell line, this loop is also involved in signal transduction mediated through the G(s) protein pathway.",
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TY - JOUR

T1 - The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to G(s)- and G(q/11)-mediated signal transduction pathways

T2 - Evidence from loop fragment transfection in GGH3 cells

AU - Ulloa-Aguirre, Alfredo

AU - Stanislaus, Dinesh

AU - Arora, Vivek

AU - Väänänen, Jeffrey

AU - Brothers, Shaun P

AU - Janovick, Jo Ann

AU - Conn, P. Michael

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N2 - The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane- proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH31' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both G(s) and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other G(s)-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH31' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH31' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the α(1B)-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach- muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the G(s) protein). Transfection of loop 3i from the D(1A) -dopamine receptor (coupled to the G(s) protein) produced a selective attenuation (40%) in G(s)-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and α(2A)-adrenergic receptor's). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH31' cell line, this loop is also involved in signal transduction mediated through the G(s) protein pathway.

AB - The GnRH receptor (GnRH-R) belongs to the rhodopsin/β-adrenergic family of G protein-coupled receptors. The intracellular domains of these receptors, particularly the regions closest to the plasma membrane in intracellular loops 2 (2i) and 3 (3i) as well as some regions located in the membrane- proximal end of the COOH-terminus, are frequently important sites for G protein coupling and specificity determination. Although studies in mouse and human GnRH-R have identified loop 2i as a critical determinant for coupling the receptor to the G(q/11)-mediated signal transduction pathway, given the functional similarity among the members of this particular G protein-coupled receptor subfamily and the fact that the GnRH-R lacks the typical intracellular COOH-terminal domain of its superfamily (a potential site for G protein coupling), we investigated the possibility that loop 3i of this receptor also participates in GnRH-R coupling to G proteins. GGH31' cells, a pituitary-derived cell line that expresses a functional rat GnRH-R coupled to both G(s) and G(q/11) proteins, were transiently transfected with a plasmid DNA containing a complementary DNA (cDNA) coding for the entire loop 3i of the GnRH-R as well as with other expression plasmids containing cDNAs encoding loop 3i of other G(s)-, G(i/o)-, or G(q/11)-coupled receptors. The effects of coexpression of these loops with the wild-type GnRH-R on inositol phosphate (IP) production, cAMP accumulation, and PRL release were then examined. Transfection of GGH31' cells with the cDNA for loop 3i of the rat GnRH-R (efficiency, 35-45%) maximally inhibited buserelin-stimulated IP turnover by 20% as well as cAMP accumulation and PRL secretion by 30%. This attenuation in cellular responses to a GnRH agonist was statistically significant (P < 0.05) compared with the responses exhibited by GGH31' cells transfected with a control plasmid and stimulated with the same GnRH agonist. Transfection of minigenes coding for loop 3i of the M1Ach-muscarinic and the α(1B)-adrenergic (G(q/11)-coupled) receptors resulted in 25-55% inhibition of maximal GnRH-evoked IP turnover. Paradoxically, loop 3i from the M1Ach- muscarinic receptor also maximally inhibited GnRH agonist-stimulated cAMP accumulation and PRL release by 40% (both effects mediated through activation of the G(s) protein). Transfection of loop 3i from the D(1A) -dopamine receptor (coupled to the G(s) protein) produced a selective attenuation (40%) in G(s)-mediated cellular responses. In contrast, receptor/G protein coupling appeared unaffected by expression of loop 3i domains derived from two receptors coupled to G(i/o) proteins (M2Ach-muscarinic and α(2A)-adrenergic receptor's). These data indicate that the third intracellular loop of the rat GnRH-R is involved in receptor G(q/11) protein coupling and/or selectivity, and in the GGH31' cell line, this loop is also involved in signal transduction mediated through the G(s) protein pathway.

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