Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus

Takashi Kusui, Mark Hellmich, Lu Hua Wang, Richard L. Evans, Richard V. Benya, James F. Battey, Robert T. Jensen

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (Mr = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-R in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used. The ability of this system to express mGRP-R in increased amounts should be useful for obtaining enough receptor for making antibodies, performing reconstitution studies, and studying GRP-R modulation by agonists, and this system also provides useful insights into the importance of both glycosylation and G protein coupling with various agonists.

Original languageEnglish (US)
Pages (from-to)8061-8075
Number of pages15
JournalBiochemistry
Volume34
Issue number25
StatePublished - 1995
Externally publishedYes

Fingerprint

Bombesin Receptors
Sf9 Cells
Baculoviridae
Insects
Glycosylation
Type C Phospholipases
Infection
GTP-Binding Proteins
Bombesin
Tissue
Oligosaccharides
Labeling
Peptide Receptors
Steroid Receptors
Molecular mass
Immunoblotting
Adenylyl Cyclases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kusui, T., Hellmich, M., Wang, L. H., Evans, R. L., Benya, R. V., Battey, J. F., & Jensen, R. T. (1995). Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus. Biochemistry, 34(25), 8061-8075.

Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus. / Kusui, Takashi; Hellmich, Mark; Wang, Lu Hua; Evans, Richard L.; Benya, Richard V.; Battey, James F.; Jensen, Robert T.

In: Biochemistry, Vol. 34, No. 25, 1995, p. 8061-8075.

Research output: Contribution to journalArticle

Kusui, T, Hellmich, M, Wang, LH, Evans, RL, Benya, RV, Battey, JF & Jensen, RT 1995, 'Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus', Biochemistry, vol. 34, no. 25, pp. 8061-8075.
Kusui, Takashi ; Hellmich, Mark ; Wang, Lu Hua ; Evans, Richard L. ; Benya, Richard V. ; Battey, James F. ; Jensen, Robert T. / Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus. In: Biochemistry. 1995 ; Vol. 34, No. 25. pp. 8061-8075.
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