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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.
Insights
Mutant cell lines showed reduced calcitonin receptor function but increased vasopressin receptor numbers. These defects are recessive and affect the same gene, suggesting a common pathway for polypeptide hormone receptor processing.
Area of Science:
- Cell Biology
- Endocrinology
- Molecular Genetics
Background:
- Mutant LLC-PK1 cell lines (FIB6, FIB5/N4) were studied to understand polypeptide hormone receptor regulation.
- Previous studies indicated altered responses to calcitonin and vasopressin in these mutants.
Purpose of the Study:
- To investigate the specific defects in calcitonin and vasopressin receptor function in FIB6 and FIB5/N4 cell lines.
- To determine the genetic basis and inheritance pattern of these receptor defects.
- To explore potential common pathways in the processing of calcitonin and vasopressin receptors.
Main Methods:
- Hormone binding assays using radiolabeled calcitonin and vasopressin.
- Measurement of adenylate cyclase and cAMP-dependent protein kinase (cAMP-PK) activation.
- Somatic cell hybridization to assess complementation of mutant phenotypes.
- Analysis of receptor expression and function in parental, mutant, and hybrid cell lines.
Main Results:
- Mutant cell lines exhibited significantly reduced calcitonin binding and activation of downstream signaling pathways.
- Concurrently, mutants showed increased vasopressin binding and receptor responsiveness.
- Somatic cell hybrids with parental cells restored normal receptor function, indicating recessive mutations.
- Hybridization between FIB6 and FIB5/N4 showed no complementation, suggesting mutation in the same gene.
- Hybridization with a 'receptorless' mutant (M18) demonstrated complementation, indicating distinct defects.
Conclusions:
- The mutations in FIB6 and FIB5/N4 are recessive and affect the same gene involved in polypeptide hormone receptor function.
- Mutant cell lines possess an increased number of functional vasopressin receptors, possibly due to compensatory mechanisms.
- The findings support a model proposing a common pathway for the processing and regulation of calcitonin and vasopressin receptors.