A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism.
Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.
Harris
Rebecca M
RM
Department of Medicine, Feinberg School of Medicine, Northwestern University, Arthur J. Rubloff Building, 420 E. Superior St., Suite 12-109, Chicago, IL 60611, USA.
Finlayson
Courtney
C
Weiss
Jeffrey
J
Fisher
Lisa
L
Hurley
Lisa
L
Barrett
Tim
T
Emge
Donna
D
Bathgate
Ross A D
RA
Agoulnik
Alexander I
AI
Jameson
J Larry
JL
D000818:Animals; D002874:Chromosome Mapping; D003456:Cryptorchidism / Q000235:genetics*; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005038:Ethylnitrosourea; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction; D013737:Testis / Q000002:abnormalities / Q000503:physiopathology
C555886:RXFP2 protein, mouse; D043562:Receptors, G-Protein-Coupled; D005038:Ethylnitrosourea