Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

Summary

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

Abstract

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood ^1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) ^1,2 and precocious puberty ⁴. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.

Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.

The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies ^1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation ⁴ as well as to alter the rate of degradation of KISS1R ⁹. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors ⁹. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Protocol

1. Site-directed mutagenesis of highly GC-rich KISS1R gene sequence

1. Template: full cDNA sequence of the human KISS1R with a Myc-tag fused to its N-terminus. This sequence is cloned into the pCS2+ expression vector, which is compatible with the mammalian cell lines subsequently used for transfections. This expression vector is referred to herein as pCS2⁺MycKISS1R.
2. Primer design: primers are designed to carry desired mutations, according to instructions of the Quikchange Site-Directed Mutagenesis kit (Stratagene). In summary:
   • Both (forward and reverse) primers must contain the desired mutation and anneal to....

Discussion

Site-directed mutagenesis has been used to study protein function by introducing nucleotide changes in the coding sequence of targeted genes for over three decades. The original technique was described in 1978 by the British-Canadian Chemist and Nobel Prize winner Michael Smith ¹⁰. Michael Smith shared the 1993 Nobel Prize in Chemistry with Kary Mullis, the American Biochemist who invented the Polymerase Chain Reaction (PCR) ¹¹. The original method described by Michael Smith has been improved ove.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x PCRx Enhancer Solution	Invitrogen	52391
PfuUltra High-fidelity DNA Polymerase Alternative Detergent	Stratagene, Agilent Technologies	600385
Dpn-I	New England Biolabs	R0176
XL10-Gold Ultracompetent E. coli cells	Stratagene, Agilent Technologies	200314
DMEM	Cellgro	10-013-CV
Fetal bovine serum	Atlanta Biologicals	S11550
Geneporter Transfection Reagent	Genlantis	T201007
Leupeptin	Calbiochem	108975
MG132	Calbiochem	47491
10xPBS	Ambion	AM9625
Protease inhibitor cocktail and PMSF	Santa Cruz Biotechnology, Inc.	Sc-24948
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific, Inc.	23225
anti-Myc tag (clone 4A6) agarose conjugate	EMD Millipore	16-219
2x loading buffer	Bio-Rad	161-0737
Criterion Tris-HCl precast gel, 4-15% gradient	Bio-Rad	345-0028
Immobilon-FL PVDF membrane	EMD Millipore	IPFL00010
Odyssey Blocking Buffer	LI-COR Biosciences	927-40000
10xTBS	Bio-Rad	170-6435
Rabbit anti-myc antibody	Cell Signaling Technology	2272
Goat anti-rabbit IRDye 800CW	LI-COR Biosciences	926-32211
Odyssey Imaging Infrared System	LI-COR Biosciences
Halt Protease Inhibitor Cocktail (100x)	Thermo Fisher Scientific, Inc.	78430