Complementation between LLC‐PK1 mutants affected in polypeptide hormone‐receptor function

David A. JANS, Thérèse J. RESINK, Brian A. HEMMINGS

Research output: Contribution to journalArticleResearchpeer-review

8 Citations (Scopus)

Abstract

The mutant LLC‐PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP‐dependent protein kinase (cAMP‐PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I‐labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half‐maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC‐PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC‐PK1 cells exhibited normal hormone binding and activation of cAMP‐PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the ‘receptorless’ mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC‐PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.

Original languageEnglish
Pages (from-to)571-576
Number of pages6
JournalEuropean Journal of Biochemistry
Volume162
Issue number3
DOIs
Publication statusPublished - 1 Jan 1987
Externally publishedYes

Cite this

@article{2c18a4400e9a4586a0f0246387314fbe,
title = "Complementation between LLC‐PK1 mutants affected in polypeptide hormone‐receptor function",
abstract = "The mutant LLC‐PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP‐dependent protein kinase (cAMP‐PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I‐labeled salmon calcitonin/mg cellular protein, which was about 1{\%} of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half‐maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC‐PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC‐PK1 cells exhibited normal hormone binding and activation of cAMP‐PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the ‘receptorless’ mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC‐PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.",
author = "JANS, {David A.} and RESINK, {Th{\'e}r{\`e}se J.} and HEMMINGS, {Brian A.}",
year = "1987",
month = "1",
day = "1",
doi = "10.1111/j.1432-1033.1987.tb10677.x",
language = "English",
volume = "162",
pages = "571--576",
journal = "European Journal of Biochemistry",
issn = "0014-2956",
publisher = "Wiley-Blackwell",
number = "3",

}

Complementation between LLC‐PK1 mutants affected in polypeptide hormone‐receptor function. / JANS, David A.; RESINK, Thérèse J.; HEMMINGS, Brian A.

In: European Journal of Biochemistry, Vol. 162, No. 3, 01.01.1987, p. 571-576.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Complementation between LLC‐PK1 mutants affected in polypeptide hormone‐receptor function

AU - JANS, David A.

AU - RESINK, Thérèse J.

AU - HEMMINGS, Brian A.

PY - 1987/1/1

Y1 - 1987/1/1

N2 - The mutant LLC‐PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP‐dependent protein kinase (cAMP‐PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I‐labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half‐maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC‐PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC‐PK1 cells exhibited normal hormone binding and activation of cAMP‐PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the ‘receptorless’ mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC‐PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.

AB - The mutant LLC‐PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP‐dependent protein kinase (cAMP‐PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I‐labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half‐maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC‐PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC‐PK1 cells exhibited normal hormone binding and activation of cAMP‐PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the ‘receptorless’ mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC‐PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.

UR - http://www.scopus.com/inward/record.url?scp=0023160786&partnerID=8YFLogxK

U2 - 10.1111/j.1432-1033.1987.tb10677.x

DO - 10.1111/j.1432-1033.1987.tb10677.x

M3 - Article

VL - 162

SP - 571

EP - 576

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 3

ER -