Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.
Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.
Lin
X
X
Oregon Regional Primate Research Center, Beaverton 97006, USA.
Janovick
J A
JA
Brothers
S
S
Blömenrohr
M
M
Bogerd
J
J
Conn
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002064:Buserelin / Q000494:pharmacology; D002397:Catfishes; D000242:Cyclic AMP / Q000096:biosynthesis; D015536:Down-Regulation / Q000235:genetics; D005786:Gene Expression Regulation; D007295:Inositol Phosphates / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011388:Prolactin / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011966:Receptors, LHRH / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D007295:Inositol Phosphates; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D011388:Prolactin; D000242:Cyclic AMP; D002064:Buserelin