Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function.

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Research Abstract Details 

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Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Abstract Text:

Beena E Thomas,Angela Wittelsberger,Iwona Woznica,Mo-Ying Hsieh,Paul Monaghan,Byung-Kwon Lee,Michael Rosenblatt,

PTHR1 mutants lacking endogenous cysteines in transmembrane and intracellular domains were generated. Mutant receptors were tested for their biological activities and mRNA and cell surface expression levels. C217 in intracellular loop 1 was determined to play a critical role in cell surface translocation and function of the receptor. INTRODUCTION: Elucidating the role of different domains of PTH receptor 1 (PTHR1) is essential for understanding the mechanism of ligand-receptor interactions. Here we present a study directed at determining the importance of cysteine residues present in the intracellular and transmembrane (TM) domains of the receptor. MATERIALS AND METHODS: Mutant receptors were generated by site-directed mutagenesis. Biological activities were characterized by adenylyl cyclase and competition binding assays. RT-PCR, ELISA, and immunofluorescence microscopy were carried out to determine receptor mRNA and protein expression levels. RESULTS: Mutations C460L and C462L in TM7, C568L in the C-terminal intracellular domain of the receptor, and removal of C397 in intracellular loop (ICL)3 by insertion of cleavage sites for Factor Xa did not affect binding affinity of PTH or agonist-induced adenylyl cyclase activity, although maximal responses (IC(max) and EC(max)) were decreased. However, mutations C217L in ICL1 or both C217L and C568L simultaneously resulted in a decrease in binding and loss of adenylyl cyclase activity. RT-PCR results showed that the observed changes in binding and activity were not caused by changes in mRNA expression. Next, we determined cell surface and total expression of the wildtype and mutant receptors by ELISA. We found that mutations of C460/C462 to L moderately decreased transfer of receptors to the cell surface. However, mutation of C217 to L in the ICL1 drastically reduced cell surface expression. Immunofluorescence and confocal microscopy studies confirmed reduced cell surface expression of receptors containing the C217L mutation. Similar results were obtained when replacing C217 and C460/C462 of the receptor with A instead of L. CONCLUSIONS: Our studies indicate that the cysteine at position 217 in ICL1 plays a critical role in translocation to the cell surface and biological function of PTHR1.

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Publishing Authors By Initials

BE Thomas,A Wittelsberger,I Woznica,MY Hsieh,P Monaghan,BK Lee,M Rosenblatt,

For similar investigative techniques: genetic techniques: gene transfer techniques: transfection research abstracts see: investigative techniques: genetic techniques: gene transfer techniques: transfection research

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Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Journal of bone and mineral research : the officia

VOLUME: 22

Page Numbers: 609-16

Journal Abbreviation: J. Bone Miner. Res.

ISSN: 0884-0431

DAY: 3

MONTH: Apr

YEAR: 2007

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 8610640

Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Keywords Mesh Terms:

KEYWORDS: Transfection

MESH TERMS: metabolism

Chemical & Substance for Abstract: Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function. Information

Substance Name: Cysteine

Registry Number: 52-90-4

Grant and Affiliation Information for Cysteine at position 217 in the intracellular loop 1 plays a critical role in human PTH receptor type 1 membrane translocation and function.

AFFILIATION: Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA.

Country: United States

AGENCY: United States NIDDK

GRANT: DK47940

ACRONYM: DK

MEDLINETA: J Bone Miner Res

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