Random mutagenesis of the third intracellular loop of the D2s dopamine receptor

Michael W. Quasney, Susan Senogles

Research output: Contribution to journalArticle

Abstract

The D2 Dopamine receptor exists in two alternate splice forms, the D2I (long) and the D2s (short). The D21 has an additional 29 amino acids in the third intracellular loop, but is otherwise identical in amino acid sequence to the D2s. We used a PCR based amplification in the presence of Mn2+ to incorporate random mutations in the third intracellular loop and C terminus of the D2s. The amplification encompassed the region from the Mrol site on the extreme N terminal region of the third intracellular loop to the PstI site located in the 3' untranslated region. The mutant receptors were assembled by cloning the PCR fragments into the MroI-Pstl sites of the wild type D2s. Thirty two mutations located throughout the third intracellular loop and cytoplasmic tail were assembled and expressed in HEK 293 cells. The mutants were assayed for T he ability to inhibit forskolin stimulated adenylyl cyclase when stimulated with agonist and for ligand binding. Several regions of the receptor were identified as being critical for coupling. The N terminal region of the third loop was critical for coupling, as mutations in this region resulted in >60% decrease in the ability of the receptor to inhibit adenylyl cyclase. Mutagenesis of the C terminal region of the third cytoplasmic yielded more complicated results, with several mutants showing increased coupling and others displaying decreased coupling to adenylyl cyclase. The amino acids close to the splice region were also extremely sensitive to random changes resulting in significant (>60%) impairment of activity.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

Fingerprint

Mutagenesis
adenylate cyclase
Dopamine Receptors
Adenylyl Cyclases
mutagenesis
mutation
Amino Acids
mutants
Mutation
receptors
Amplification
Polymerase Chain Reaction
forskolin
amino acids
Dopamine D2 Receptors
HEK293 Cells
Cloning
3' Untranslated Regions
3' untranslated regions
Colforsin

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Random mutagenesis of the third intracellular loop of the D2s dopamine receptor. / Quasney, Michael W.; Senogles, Susan.

In: FASEB Journal, Vol. 12, No. 8, 01.12.1998.

Research output: Contribution to journalArticle

@article{52f7e78d3ad3456ca5e7d900ff1d8b90,
title = "Random mutagenesis of the third intracellular loop of the D2s dopamine receptor",
abstract = "The D2 Dopamine receptor exists in two alternate splice forms, the D2I (long) and the D2s (short). The D21 has an additional 29 amino acids in the third intracellular loop, but is otherwise identical in amino acid sequence to the D2s. We used a PCR based amplification in the presence of Mn2+ to incorporate random mutations in the third intracellular loop and C terminus of the D2s. The amplification encompassed the region from the Mrol site on the extreme N terminal region of the third intracellular loop to the PstI site located in the 3' untranslated region. The mutant receptors were assembled by cloning the PCR fragments into the MroI-Pstl sites of the wild type D2s. Thirty two mutations located throughout the third intracellular loop and cytoplasmic tail were assembled and expressed in HEK 293 cells. The mutants were assayed for T he ability to inhibit forskolin stimulated adenylyl cyclase when stimulated with agonist and for ligand binding. Several regions of the receptor were identified as being critical for coupling. The N terminal region of the third loop was critical for coupling, as mutations in this region resulted in >60{\%} decrease in the ability of the receptor to inhibit adenylyl cyclase. Mutagenesis of the C terminal region of the third cytoplasmic yielded more complicated results, with several mutants showing increased coupling and others displaying decreased coupling to adenylyl cyclase. The amino acids close to the splice region were also extremely sensitive to random changes resulting in significant (>60{\%}) impairment of activity.",
author = "Quasney, {Michael W.} and Susan Senogles",
year = "1998",
month = "12",
day = "1",
language = "English (US)",
volume = "12",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "8",

}

TY - JOUR

T1 - Random mutagenesis of the third intracellular loop of the D2s dopamine receptor

AU - Quasney, Michael W.

AU - Senogles, Susan

PY - 1998/12/1

Y1 - 1998/12/1

N2 - The D2 Dopamine receptor exists in two alternate splice forms, the D2I (long) and the D2s (short). The D21 has an additional 29 amino acids in the third intracellular loop, but is otherwise identical in amino acid sequence to the D2s. We used a PCR based amplification in the presence of Mn2+ to incorporate random mutations in the third intracellular loop and C terminus of the D2s. The amplification encompassed the region from the Mrol site on the extreme N terminal region of the third intracellular loop to the PstI site located in the 3' untranslated region. The mutant receptors were assembled by cloning the PCR fragments into the MroI-Pstl sites of the wild type D2s. Thirty two mutations located throughout the third intracellular loop and cytoplasmic tail were assembled and expressed in HEK 293 cells. The mutants were assayed for T he ability to inhibit forskolin stimulated adenylyl cyclase when stimulated with agonist and for ligand binding. Several regions of the receptor were identified as being critical for coupling. The N terminal region of the third loop was critical for coupling, as mutations in this region resulted in >60% decrease in the ability of the receptor to inhibit adenylyl cyclase. Mutagenesis of the C terminal region of the third cytoplasmic yielded more complicated results, with several mutants showing increased coupling and others displaying decreased coupling to adenylyl cyclase. The amino acids close to the splice region were also extremely sensitive to random changes resulting in significant (>60%) impairment of activity.

AB - The D2 Dopamine receptor exists in two alternate splice forms, the D2I (long) and the D2s (short). The D21 has an additional 29 amino acids in the third intracellular loop, but is otherwise identical in amino acid sequence to the D2s. We used a PCR based amplification in the presence of Mn2+ to incorporate random mutations in the third intracellular loop and C terminus of the D2s. The amplification encompassed the region from the Mrol site on the extreme N terminal region of the third intracellular loop to the PstI site located in the 3' untranslated region. The mutant receptors were assembled by cloning the PCR fragments into the MroI-Pstl sites of the wild type D2s. Thirty two mutations located throughout the third intracellular loop and cytoplasmic tail were assembled and expressed in HEK 293 cells. The mutants were assayed for T he ability to inhibit forskolin stimulated adenylyl cyclase when stimulated with agonist and for ligand binding. Several regions of the receptor were identified as being critical for coupling. The N terminal region of the third loop was critical for coupling, as mutations in this region resulted in >60% decrease in the ability of the receptor to inhibit adenylyl cyclase. Mutagenesis of the C terminal region of the third cytoplasmic yielded more complicated results, with several mutants showing increased coupling and others displaying decreased coupling to adenylyl cyclase. The amino acids close to the splice region were also extremely sensitive to random changes resulting in significant (>60%) impairment of activity.

UR - http://www.scopus.com/inward/record.url?scp=33749116645&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33749116645&partnerID=8YFLogxK

M3 - Article

VL - 12

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 8

ER -