M elanocyte Stim ulating H orm one-Like(MSH-Like) Activity
T E R R Y O . M A T S U N A G A , * V I C T O R J . H R U B Y , .1 M I C H A L L E B L ,i "
A N A M A R I A D E L A U R O C A S T R U C C I: ~ A N D M A C E . H A D L E Y §
Departments o f *Chemistry and §Anatomy, University of Arizona, Tucson, AZ 85721"PDepartment of Chem istry, Institute o f Organ ic Chem istry and Biochem istry
Czechoslovak Acade my of Sciences, P rague 6, CS166 10, Czechoslovakia
$'Departamento de Fisiologia, lnstituto de BiocienciasUniversidade de Sao Paulo C P I 1176, Sao Paulo, 05499, Brazil
R e c e i v e d 6 F e b r u a r y 1 9 8 9
MATSUNAG A, T. O. , V. J . HRUBY, M. LEBL, A. M. DE LAURO CASTRUCCI AND M. E. HADLEY. Melanin concentratinghormone (MC H): Structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity. PEPTIDES 10(4)773-778, 198 9.--M elani n concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-C~s-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-C)/s-Trp-Glu-Val, synthesized in the brain and secreted from the p ars nervosa of teleost fish. This hormone stimulatesmelanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melansomedispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH whichis responsible for its MSH-like component of activity. Remov al of the N-terminal amino acid results in an almost total loss of M SH-likeactivity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, M CH (I-16 ), is about 100 times
less active than M CH. Therefore, the entire heptadecapeptide sequence of M CH appears to contribute to the M SH-like activity ofMCH . Ring-contracted analogues (e.g., [AlaS,Cyst°]MCH) of MCH are almost devoid of any melanosome aggregating (MC H-like)activity bu t generally possess considerable or as great an M SH-like activity as MC H. Racemization of M CH b y heat-alkali treatmentdrastically reduces the MCH-like activity of MCH , bu t does not enhance the M SH-like activity of the hormone.
M A N Y s p e c i e s o f f r o g s , l i z a r d s , a n d f i s h a r e d e p e n d e n t u p o nh o r m o n a l l y - i n d u c e d c h r o m a t i c r e s p o n s e s f o r t h e i r e x i st e n c e a n d
s u r v i v a l. T w o h o r m o n e s , m e l a n o c y t e c o n c e n t ra t i n g h o r m o n e ( M C H )a n d e t - m e l a n o c y t e s t i m u l a t in g h o r m o n e ( e t - M S H ) , a r e p r i m a r i l y
r e s p o n s i b l e f o r i n d u c i n g s k i n l i g h t e n i n g a n d s k i n d a r k e n i n g ,r e s p e c t i v e l y . M C H s t i m u l a t e s m e l a n o s o m e a g g r e g a t i o n w i t h i n
m e l a n o c y t e s o f t e l e o s t f i s h , a n d e t - M S H s t i m u l a t e s m e l a n o s o m e
d i s p e r s i o n w i t h i n m e l a n o c y t e s o f m o s t v e r t e b r a t e s p e c ie s ( 1 , 5 , 8 ,10 , 13 , 14) . MCH i s a he p ta de c a pe pt ide w i th a c e n t r a l ly loc a te ddi su l f ide br idge be tw e e n r e s idue s 5 a nd 14 (F ig . 1 ) .
I t h a s b e e n d e m o n s t r a t e d t h a t M C H c a n a l s o p r o m o t e t h ed i s p e r s io n ( M S H - l i k e a c t i v i ty ) o f m e l a n s o m e s i n b o t h t h e f r o g a n dl i za r d s k i n b i o a ss a y s ( 2 1 ,2 2 ) . T h e M S H - l i k e a c t i v i ty o f M C H h a sb e e n c o n f i r m e d b y o t h e r l a b o r a t o r i e s a s w e l l ( 7 , 1 2 ) . R e c e n t l y ,
r i n g - c o n t ra c t e d M C H a n a l o g u e s w e r e f o u n d t o e x h i b i t d r a s t i c al l y
r e d u c e d o r n o M C H a c t i v i t y ( 1 6 ) . I n t e r e s t i n g l y , h o w e v e r , s o m e
a na logue s s t i l l r e t a ine d MSH - l ike a c t iv i ty (16) . Be c a use of the i r
dua l a c t iv i t i e s , Ca s t ruc c i a nd c o-w orke r s (6) ha ve sugge s te d ap o s s i b l e e v o l u t i o n a r y l i n k b e t w e e n t h e t w o h o r m o n e s . T h u s , f r o m
a c o m m o n s e q u e n c e , m u t a t i o n s c o u l d h a v e r e s u l t e d i n t w odi f f e re n t pe p t ide s w i th d i f f e re n t b io log ic a l func t ions .
W e h a v e s y n t h e s i z e d a n e n t i r e s e r i e s o f M C H f r a g m e n t
a n a l o g u e s c o n s i s t i n g o f s e q u e n t i a l d e le t i o n s o f e i t h e r o r b o t h t h eN - a nd C- te rm in i . A l l o f the se t runc a te d a na logue s s t i l l pos se s se dthe in ta c t c e n t r a l c yc l i c d i su l f ide br idge s t ruc ture of MCH . In a
p r e v i o u s p u b l i c a t i o n w e r e p o r t ed o n t h e M C H - l i k e a c t i v i ti e s o ft h e s e f r a g m e n t a n a l o g u e s a s d e t e r m i n e d i n f i s h s k i n b i o a s s a y s( 1 8 ) . W e c o n c l u d e d t h a t t h e f r a g m e n t a n a l o g u e , M C H ( 5 - 1 5 ) , i st h e m i n i m a l s e q u e n c e f o r e q u i p o t e n c y t o M C H i n t h e f i s h s k i n
a s sa y ( e e l , Synbranchus marmoratus). In the pre se n t r e por t w e
~Requests for reprints should be addressed to V ictor J. H ruby.
Ae-Ser-Tyr-Ser-Met -G -Hi s-Phe-Azg-Tr p-Gly-Lys-Pr o-Va i N~
" - - ,mM i L ~ - ~ t m ~ t z i t t n g ~ ( l O l l )
A s p -T h r - M e t - ~ - V a 1 G y- A r - V a I T y r A t - P ~ o - C ~ - T r p - G 1 - v a I
FIG. 1. Primary structures of melanin concentrating hormone (M CH ) and
melanocyte stimulating hormone (¢t-MSH).
d e t e r m i n e d t h e M S H - l i k e a c t i v i t ie s o f t h e a n a l o g u e s a s d e t e r m i n e d
i n t h e f r o g a n d l i z a r d s k i n b i o a s s a y s . T h e r e s u l ts r e v e a l s o m e o f t h e
s t r u c t u r a l a s p e c t s o f M C H r e l a t e d t o i t s M S H c o m p o n e n t o f
m e l a n o t r o p i c a c t i v it y .
METHODS
A l l p e p t i d e s w e r e s y n t h e s i z e d u s i n g a m o d i f i c a t i o n o f t h e
m e t h o d o f L e b l a n d c o - w o r k e r s ( 1 6 ) , a s d e s c ri b e d b y M a t s u n a g a e t
a l . ( 18) . I t s hou ld be no ted tha t s t r i c t a t t en t ion was pa id to
a v o i d i n g a n y c o n t a c t w i t h ¢ t - M S H o r a n y p o t e n t ( x - M S H a n a -
l o g u e s . T h i s n e c e s s i t a t e d t h o r o u g h w a s h i n g o f a l l g l a s s w a r e w i t h
b a s e f o l l o w e d b y a n a c i d w a s h , a n d t h e n r i n s i n g w i t h d i s t i ll e dH : O . A l l c o l u m n s u t i li z e d w e r e f r e e o f a n y e x p o s u r e t o o t - M S H o r
r e l a te d p e p t i d e s . A s a n a d d e d p r e c a u t i o n , s e l e c t e d p e p t i d e s w e r e
s y n t h e s i z e d i n a l a b o r a t o r y t o t a l l y d e v o i d o f a n y s y n t h e s i z e d
m e l a n o t r o p i n s .
F r o g s k i n a s s a y s w e r e b a s e d u p o n p r e v i o u s w o r k d e s c r i b e d i n
the l i t e r a tu r e ( 3 , 1 l , 19 ). R a n t , p i p i e n s , a s pec ies o f f r og na t ive to
N o r t h A m e r i c a n r e g i o n s , w e r e o b t a i n e d f r o m K o h n ' s S c i e n t i f i c
S u p p l y C o m p a n y , C r e rm a n t o w n , W I . A n i m a l s w e r e s a c r if i c e d b y
d e c a p i t a t i o n a n d t h e l e g a n d t h i g h s k i n s w e r e r e m o v e d a n d k e p t
v i a b l e i n a b a t h o f p h y s i o l o g i c a l s a l i n e ( R i n g e r ) s o l u t i o n . S k i n s
w e r e s t r e t c h e d o v e r P V C r i n g s a n d t h e n b a s e l i n e l i g h t r e f l e c ta n c e
w a s m e a s u r e d v i a a P h o t o v o l t ( m o d e l 6 7 0 ) r e f l e c t o m e t e r . W h e n
s t i m u l a t e d b y m e l a n o t r o p i n s , m e l a n s o m e s a r e i n d u c e d t o m i g r a t e
f r o m t h e p e r i n u c l e a r s p a c e t h r o u g h o u t t h e d e n d r i t i c p r o c e s s e s o f
m e l a n o c y t e s . T h i s l e a d s t o a g e n e r a l i z e d d a r k e n i n g o f t h e e n t i r es k i n . S i n c e s k i n d a r k e n i n g r e s u l t s i n a n i n c r e a s e i n l i g h t a b s o r -
b a n c e , r e f l e c t a n c e m e a s u r e m e n t s y i e l d a d e c r e a s e i n r e f l e c t a n c e .
U p o n r e m o v a l o f t h e m e l a n o t r o p i n ( i . e . , b y r i n s i n g w i t h R i n g e r
s o l u t i o n ) a r e a g g r e g a t i o n o f m e l a n o s o m e s t o t h e p e r i n u c l e a r s p ac e
o c c u r s , a n d t h e s k i n s g e n e r a l l y r e l i g h te n t o t h e i r o r i g i n a l b a s e l i n e
v a l u e s .
R a c e m i z a t i o n s t u d i e s w e r e c o n d u c t e d a c c o r d i n g t o t h e p r o c e -
d u r e s o f B o o l e t a l . ( 4 ) and E ber l e e t a l . ( 7 ) . S k i n d a r k e n i n g w a s
d e t e r m i n e d v i a p h o t o r e f l e c ta n c e a s a b o v e . A l l d a t a w e r e c o m p i l e d
a n d a n a l y z e d b y p l o t t i n g t h e p e r c e n t s k i n d a r k e n i n g v s . - l o g
c o n c e n t r a t i o n .
RESULTS
M e l a n i n c o n c e n t r a t in g h o r m o n e ( M C H ) s t im u l a t es m e l a n o -
s o m e a g g r e g a t i o n w i t h i n t e l e o s t m e l a n o c y t e s b u t n o t w i t h i n
p i g m e n t c e l l s o f t e t r a p o d s ( f r o g s a n d l i z a r d s ) . R a t h e r , M C H
e x h i b i t s M S H - l i k e a c t i v i t y w h e n u s e d a t h i g h c o n c e n t r a t i o n s i n
t e t ra p o d s ( F i g . 2 ) . W e p r e v i o u s l y d e m o n s t r a t e d t h a t M C H , a s w e l l
a s t h e f r a g m e n t a n a l o g u e , M C H ( I - 1 4 ) , p o s s e s s e d M S H - l i k e
a c t i v i t y w h e r e a s M C H ( 5 - - 1 7 ) a n d t h e c e n t r a l c y c l i c s e q u e n c e ,
M C H ( 5 - 1 4 ) , d i d n o t . T h i s s u g g e s t e d t h a t t h e N - t e r m i n a l 1 . - 4
s e q u e n c e , o r s o m e c o m p o n e n t t h e r e o f , w a s r e s p o n s i b l e f o r t h e
M S H - l ik e a c t i v it y o f M C H . S y n t h e s is o f M C H a n a l o g u e s w it h
N - t e r m i n a l d e l e t i o n s r e v e a le d t h a t r e m o v a l o f t h e t e r m i n a l a m i n o
ac id r es u l t ed in a near t o t a l l o s s o f M SH- l ike ac t iv i ty r e l a t ive to
M C H . M C H ( 2 - 1 7 ) , M C H ( 3 - 1 7 ) a n d M C H ( 6 - 1 7 ) e x h i b it e d g r e at l y
0 to
~ g
7 0 '
6O
5O
4 0
3 0
20 1
. ~ n o
~ - i o l
• ~ ' -MSH
• M CH
0 C o n t r o l
/
/l i
/ JI I
= /
t
/ / [ I I I I I
I I I0 9 8 7 6
- Log r c onc en t r o t i on ] ( M )i . . J
FIG. 2. In vitro demonstration of the M SH-like activity of MCH as seen inthe frog ( R . p i p i e n s ) skin bioassays. Each value is the m ean, _ S.E.,darkening response of the skins to a-M SH (N = 10) or MCH (N = 5) at theconcentrations indicated.
d i m i n i s h e d M S H - l i k e a c t i v i t y i n b o t h t h e f r o g a n d t h e l i z a rd ( d a t a
n o t s h o w n ) s k i n b i o a s s a y s ( F i g . 3 ) . A t v e r y h i g h c o n c e n t r a t i o n s ,
t h e s e M C H a n a l o g u e s w e r e f u l l M S H - l i k e a g o n i s t s a l t h o u g h th e y
w e r e m o r e t h a n 1 0 0 - f o l d l e s s a c t i v e th a n M C H ( F i g . 3 ) . A l t h o u g h
t h e m e l a n o t r o p i c a c t i v i t y o f M S H i s r a p i d l y r e v e r s e d f o l l o w i n g
5 0
4 0! i =IO
• MCH
A M C ~ _ ~
• MCH3.11,
0 MCH4. 17
• ~CHe.~7
II0
/
!I I I I I
9 8 7 6 5
FIG. 3. MCH fragment analogues minus the first [MCH(2-17)]0 second[MCH(3-17)], third [MCH(4-17)], or fourth [MCH(5-17)] amino acidresidues of the N-terminus lack of MSH-iike activity. Each value is themean, -+-S.E., darkening response of frog skins (N= 10) to the m elano-tropins at the concen trations noted.
rinseI 1 I 1 t l i t I I I0 30 60 90 120 150""180 10 240
T,me (minutes)
FIG. 4. In vitro demonstration that N-terminal deleted fragment analogues
of MCH have greatly reduced MS H-like potency. Following transfer of theskins to fresh Ringer in the absence of the peptides (Ringer rinse), the skinscontinued to remain darkened in response to M CH but not to the fragmentanalogues even over a long period of time. Each value is the mean, - S.E .,darkening response of frog skins (N =6 ) to the melanotropins (10 -6 M).
t r a n s f e r o f f r o g o r l i z a r d s k i n s t o m e d i u m w h i c h l a c k s t h e
h o r m o n e , t h e M S H - l i k e a c t i v i ty o f M C H i s o f t e n p r o lo n g e d i n
e i t h e r s p e c i e s w h e n u s e d a t h i g h c o n c e n t ra t i o n s ( F i g . 4 ) . H o w e v e r ,
5 0
4 0
¢._¢
I0
V I M ~ l ' l~ . a t /
• MCHt.~
/ / / II l I I I I
I0 9 8 7 6 5
- ~ ~ o , ~ , ~ ] (M )
T A B L E 1
RELATIVE POTENCIESOF MCH FRAGMENTANALOGUESASDETERMINED BY THE IN V ITRO FISH (SYNBRANCHUS MARMORATUS)
AND FROG (RANA PIPIEN5)SKIN BIOASSAYS
Fish Skin Bioassay Frog Skin Bioassay
PEPTIDE Potency Relative to MCH Potency Relative to MSH
M C H ( I - 1 7 )
MCH(2-17)
MCH(3-17)
MCH(4-17)
MCH(5-17)
M C H ( I - I 6)
MCH(2-16)
MCH(3-16)
MCH(4-16)
MCH(5-16)
M C H ( I - 1 5 )
MCH(2-15)
MCH(3-15)
MCH(4--15)
MCH(5-15)
MCH(1-14)
MCH(2-14)
MCH(3-14)
MCH(4-14)
MCH(5-14)
1.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
1.0
1.0
1.0
1.0
1.0
0. 1
0.07
0.05
0.023
0.014
0.001
0.000005
0.000005
0 . 0 0 0 0 0 5
(17) <0.000001
0.00003
0.000005
0.00004
0.00001
0.000007
0.00003
0.00001
0.00001
0.000004
0.000004
[0.07 (17)] 0.000006
0.000005
<0.000001
<0.000001
[0.003(17)] <0.000001
ev en o v e r a l o n g p e r i o d o f in cu b a t i o n t h e se an a l o g u es fa i l ed t o
e x h i b i t p ro l o n g e d a c t i v i ty o n f r o g m e l a n o c y t e s ( F i g. 4 ) . M C H , a s
i t h ad d o n e p rev i o u s l y , ma i n t a i n ed i t s p ro l o n g ed ac t i v i t y (F i g . 4 ) .
M o s t f r a g m e n t a n a l o g u e s o f M C H , l i ke t he p a r e nt h o r m o n e ,
p o s s e s s e d s o m e M S H - l i k e a c t i v i t y w h e n u s e d a t h i g h c o n c e n t r a -
t i on s ( e . g . , 1 0 - 5 M ) . R e m o v a l o f t h e C - t e r m i n a l a m i n o a c i d , V a l ,
f r o m M C H r e s u l te d in a p e p t i de , M C H ( I - 1 6 ) , w h i c h e x h i b i t e dab o u t ' /t oo t h e MSH -l i k e ac t i v i t y o f M CH (Fi g . 5 ) . Th e sh o r t e r
C - t e rm i n a l d el e te d f r ag m e n t a n a lo g u e s , M C H ( 1 - 1 5 ) a n d M C H ( I -
1 4 ), a l so ex h i b i t ed a s i mi l a r d i mi n i sh ed M SH -l i k e ac t i v i t y (F i g . 5 ;
T a b l e 1 ); M C H ( I - 1 4 ) s h o w i n g t h e m o r e dr a s ti c d e c r e a s e in
a c t i v it y ( T a b l e l ). T h u s , r e m o v a l o f r e s i d u e s f r o m e i t h e r t h e a m i n o
o r c a r b o x y t e r m i n i l e d t o r e d u c t io n s o f m e l a n o t r o p i c a c t i v i t y o f a t
l e a st t w o o r d e r s o f m a g n i t u d e r e l a t i v e t o M C H . O n e m a y n o t e t h a t
i n Tab l e l , t h e m o s t a c t i v e f rag m en t an a l o g u e i s b ut %o .0 0o t h e
p o t e n c y o f c t - M S H . E v e n u s i n g t h e m o s t m e t i c u l o u s c a r e , o n e
can n o t re l i ab l y a s se ss t h e p u r i t y o f a samp l e ( f ro m co n t ami n a t i o n )
b ey o n d t h i s l i mi t u s i n g cu r ren t p u r i f i c a t i o n sch emes . H en ce , t h e
v e ry l o w i n v i t ro p o t en cy may b e an i n d i ca t i o n t h a t t h e se
a n a l o g u e s c o u l d b e e v e n l e s s p o t e n t o r e v e n c o m p l e t e l y i n a c t iv e .
T h e m e l a n o t r o p i c a c t i v it y o f c t - M S H ( a n d I 3 -M S H ) i s e n h a n c e d
b y h ea t -a l k a l i t r e a t men t (F i g . 6 ) (4 ) . H ea t -a l k a l i t r e a t men t o fc t -MSH a l so l e ad s t o a p ep t i d e t h a t ex h i b i t s p ro l o n g ed ( re s i d u a l )
ac t i v i t y (F i g . 6 ) . A recen t rep o r t h a s su g g e s t ed a s i mi l a r re su l t
u p o n h e a t - a l ka l i t re a t m e n t o f M C H ( 7 ). W e h a v e r e p e a t e d t h e
ex p e r i men t an d h av e fo u n d t h a t h ea t -a l k a l i t r e a t men t d i d n o t
e n h a n c e t h e m e l a n o t r o p i c a c t i v it y o f M C H . I n f a c t, w e o b s e r v e d i n
sev e ra l ex p e r i men t s a mo d es t red u c t i o n i n p o t en cy (F i g . 7 ) .
FIG. 5. In vitro demonstration that C-terminal deleted fragment analogueslacking C-terminal amino acids also lose considerable (compared to MC H)MSH -like potency. Each value is the mean, _+ S.E ., darkening response o flizard skins (N= 6) to the melanotropins (10 -6 M).
DISCUSSION
T h e s e r e s u l t s c l e a r l y d e m o n s t r a t e t h a t M C H p o s s e s s e s t w o
c o n t r a s ti n g m e l a n o t r o p ic a c t i v i t ie s . T h e h o r m o n e s t i m u l a te s m e l -
FIG. 6. In vitro demonstration that heat-alkali treatment (racemization) ofot-MSH leads to enhanced a nd prolonged biological activity. Ea ch value isthe mean, ± S.E.. darkening response of frog skins (N = 6) to a-MSH andto racemized a-M SH (10 - no M).
a n o s o m e a g g r e g a t i o n a t p h y s i o l o g i c a l c o n c e n t r a t i o n s i n t e l e o s t
f i s h e s a n d a t h i g h e r c o n c e n t r a t i o n s s t i m u l a t e s m e l a n o s o m e d i s p e r -
s i o n w i t h i n m e l a n o c y t e s o f f i s h e s , f r o g s a n d l i z a rd s .
O u r o r i g i n a l r e su l t s d e m o n s t r a t e d t h a t s o m e c o m p o n e n t o f th e
N - t e r m i n a l t e tr a p e p t id e t a i l o f M C H w a s n e c e s s a r y f o r M S H - l i k e
a c t i v i t y ( 9 ) . T h a t o r i g i n a l o b s e r v a t i o n w a s s u p p o r t e d b y o u r
p r e s e n t s t u d y . R e m o v a l o f t h e N - t e r m i n a l a m i n o a c i d , a s p a r t i c
a c i d , r e s u l t e d i n a n a n a l o g u e w i t h a n a l m o s t t o t a l l a c k o f M S H - l i k e
a c t i v i t y r e l a t iv e t o M C H ( a t 1 0 - 6 M ) i n b o t h t h e f r o g a n d l i z a rds k i n b i o a s s a y s . N o t s u r p r i s in g , t h e r e fo r e , w a s t h e o b s e r v a t i o n t h a t
5 0
4 O
I0
• & - M S H
/• Ro ce m ize d CH0 C o ntro l / / ~
I I I I I
9 8 7 6 5
/e5
d . I I
io
FIG. 7. In vitro demonstration that heat-alkali treatment of MCH do es notenhance the MSH-like activity of the pcptide. Each value is the mean.
.4 -_ S.E., darkening of the skins (N = 6) at the concentrations noted.
t h e M C H ( 3 - 1 7 ) , M C H ( 4 - - 1 7 ) a n d M C H ( 5 - 1 7 ) a n a l o g u e s a l s o
s h o w e d d e c r e a s e d M S H - l i k e a c t i v i t y ( F i g . 2 ) . R e m o v a l o f t h e
C - t e r m i n a l a m i n o a c i d r e s i d u e a l s o r e s u l t e d i n a d r a m a t i c ( 1 0 0 -
f o l d) l o s s o f M S H - l i k e a c t i v i t y c o m p a r e d t o M C H ( T a b l e 1 ). T h e
C - t e r m i n a l tr u n c a t e d p e p ti d e s , M C H ( 1 - 1 4 ) a n d M C H ( 1 - 1 5 ) , w e r e
e v e n l e s s p o t e n t t h a n M C H ( I - 1 6 ) . T h e s e r e s u l t s , t a k e n t o g e t h e r ,
s u g g e s t t h a t t h e e n t i r e h e p t a d e c a p e p t i d e s e q u e n c e o f M C H c o n -
t r i b u te s i n s o m e w a y t o t h e M S H - l i k e p o t e n c y o f t h e h o r m o n e .
A l t h o u g h t h e N - t e r m i n a l a m i n o a c i d r e s i d u e w a s e s s e n t i a l fo r
t h e f u l l M S H - l i k e a c t i v i t y o f M C H , a l l a n a l o g u e s , w h e t h e r
t r u n c a t e d a t t h e N - o r C - t e r m i n i , o r b o t h , e x h i b i t e d s o m e s k i n
d a r k e n i n g ( m e l a n o s o m e d i s p e r s i o n ) w h e n u s e d a t v e r y h i g h
c o n c e n t r a t i o n s ( i . e . , 1 0 - 5 M ) . T h i s w a s m o s t d r a m a t i c a l l y d e m -
o n s t r a t e d b y L e b l a n d c o - w o r k e r s w i t h a s e r ie s o f r i n g - c o n t r a c t e d
a n a l o g u e s ( 1 6 ) . T h e m o s t p o t e n t o f t h e s e l a t t e r a n a l o g u e s w a s t h e
ana logue [ C ~, s T ] M C H( 7- 17) . T h i s pep t ide , pa r t i cu l a r ly i n t he
l i za r d s k in b ioas s ay , pos s es s ed the g r ea t es t M SH- l ike ac t iv i ty ,
b e i n g a b o u t 1 0 t i m e s m o r e p o t e n t i n t h a t r e sp e c t t h a n M C H i t s e lf
( 1 7 ). H e n c e , m a n i p u l a t i o n o f r i n g s i z e h a s a p p a r e n t l y r e su l t e d i n
conf or mat iona l and /o r t opogr aph ica l f ea tu r es t ha t a r e l es s f avor -
a b l e f o r M C H - l i k e a c t iv i t y b u t , r a th e r , m o r e c o n d u c i v e t o s t i m u -
l a t i n g M S H - l i k e a c t i v i ty .
M a n y y e a r s a g o i t w a s d e m o n s t r a t e d t h a t h e a t - a l k a li t r e a tm e n t
o f a - M S H l e d to s e v e r a l p h e n o m e n a r e l a ti v e t o th e m e l a n o t r o p i c
ac t iv i ty o f t he pep t ide : I ) r e t a r da t ion o f r es pons e , 2 ) enhanced
p o t e n c y , a n d 3 ) p r o l o n g e d a c t i v i t y ( 4 , 1 0 ) . S i n c e M C H e x h i b i t s
M S H - l i k e a c t i v i t y , t h e q u e s t i o n a r o s e a s t o w h e t h e r s i m i l a r
c h a n g e s c o u l d b e i n d u c e d f o l l o w i n g h e a t - a l k a li t re a t m e n t o f M C H .
A l t h o u g h t h e M C H - l i k e a c t i v i t y w a s d r a s t ic a l l y re d u c e d ( d a t a n o t
s h o w n ) , n o e n h a n c e m e n t o f t h e M S H - l i k e a c t i v i ty o f th e h o r m o n e
c o u l d b e e f f e c t e d i n M C H o r s e v e r a l o f i t s a n a l o g u e s . T h e s e
o b s e r v a t i o n s w e r e c o n s i s t e n t l y o b s e r v e d . T h e r e f o r e , w e d o n o t
s u p p o r t th e c l a i m ( 7 ) t h a t th e M S H - l i k e a c t i v i t y o f M C H c o u l d b e
e n h a n c e d b y h e a t - a l k a l i t r e a t m e n t . I n f a c t , w i t h r e s p e c t t o o u r
e x p e r i m e n t s , t h e M S H - l i k e a c t i v i t y o f M C H w a s m o d e s t l y d i m -
in i s hed .
I n s e v er a l o th e r c o m m u n i c a t i o n s w e h a v e c o m m e n t e d o n t h e
p o s s i b l e e v o l u t i o n a r y re l a t i o n s h i p s o f c x -M S H a n d M C H . I t
a p p e a r s t h a t t h e m e l a n o t r o p i c a c t i v i t y ( m e l a n o s o m e a g g r e g a t i o n )o f M C H i s r es t r i c t ed to t e l eos t f i s hes and the i r immedia t e
f o r b e a r e r s ( H o l o s t e a n f i s h e s ) , a - M S H , o n t h e o t h e r h a n d , r e g u -
l a t e s m e l a n o s o m e d i s p e r s i o n a n d / o r m e l a n i n s y n t h e s i s i n a l l
s p e c i e s o f v e r t e b r a t e s ( i n c l u d i n g c a r t il a g i n o u s f i sh e s a n d h u m a n s ) .
T her e f o r e , s ince the bony f i s hes wer e an ea r ly o f f - s hoo t f r om the
m a i n s t e m o f v e r te b r a t e e v o l u t i o n , i t m i g h t b e a s s u m e d t h a t M S H
is ances t r a l t o M C H.
S i n c e a n M C H - l i k e p e p t i d e i s c l a i m e d t o b e p r e s e n t w i t h i n t h e
m a m m a l i a n ( i n c l u d i n g h u m a n ) b r a i n (2 3 ) , a n M C H - l i k e p e p t i d e
m u s t h a v e b e e n p r e s e n t b e f o r e t h e b o n y f i s h e s e v o l v e d a s a n
o f f - sh o o t o f t h e m a i n l i n e v e rt e b r a te e v o l u t i o n . S i n c e M C H l a c k s
M C H - l i k e a c t i v i t y i n a l l m e m b e r s o f t h e t e t r a p o d l i n e , t h e n i t
r e m a i n s t o b e d e t e r m i n e d w h e t h e r M C H o r a r e la t e d p e p t id e p l a y s
s ome o ther phys io log ica l r o l e i n ver t eb r a t es .
B e c a u s e o f i t s a b i l i t y t o c a u s e m e l a n o s o m e d i s p e r s i o n / a g g r e -g a t i o n , i t i s c o n c e i v a b l e t h a t M C H m a y h a v e e v o l v e d b y g e n e
d u p l i c a t i o n o f a m e l a n o t r o p i n ( e . g . , c x- o r 1 3 -M S H , o r A C T H )
f o l l o w e d b y m u t a t i o n s i n o n e o f t h e r e d u n d a n t M S H c o d o n s . O n
t h i s n o t e , w e h a v e c o m p a r e d t h e m R N A s e q u e n c e s f o r b o t h
o t - M S H a n d M C H ( 2 0 ) ( F i g . 8 ) . A l t h o u g h w e c o u l d n o t r e a d i l y
f i n d a n y s i g n i f i c a n t a m o u n t o f p r i m a r y s e q u e n c e h o m o l o g y e i t h e r
f r o m s e q u e n c e c o m p a r i s o n , f r o m c h a n g e s d u e t o p o s s i b l e f r a m e -
s h i ft m u t a t i o n s , o r f r o m r e s i d u e d e l e t i o n s , o n e p o s s i b l y i n t e r e s ti n g
c o m p o n e n t d i d e m e r g e . I n a - M S H , s t r u c tu r e - a c t iv i t y r e l a t io n s h i p s
h a v e s u g g e s t e d t h e i m p o r t a n c e o f P h e T - A rg~ ( 10) t o i t s b io log ic a l
p o t e n c y. I n M C H , M a t s u n a g a e t a l . . h a v e s u g g e s t e d t h e i m p o r -
t a n c e o f t h e T y r t t - A r g l 2 - P r o ~3 r e s id u e s o f M C H . I n t e r e s t i n g l y . a
S T R U C T U R E - F U N C T I O N A S P E C T S O F M C H 777
IJ . m - n i d w ~
Bet Tyr f J e r M e t u l u M|s Phe A r g T r i p
UCt l UAU UCU AUG GA& CAU uLqJ CGU UGG
U l y L y s P r o V a l
GG D A A A C C O G U l l
5 N e l l . a u
A s p T y r N e t , A r g C y s N e t V a l U l y & r g V a l T y r A r g
G A U A C A A U G C G U U G U A U G G ~ J G G U C G U G Ul l U A U C(RJ
C G U C C C C C C C C
U A A A A A A A
C G G G G G U
c y s T r p G l u V a l
C u C
g
G
FIG. 8. M essenger RNA (mRN A) sequences of et-MSH and MCH.
p o i n t mu t a t i o n o f u rac i l t o ad en i n e fo r t h e Ph e co d o n w o u l d
c o n v e r t t h e P h e t o a T y r ( F i g . 8 ) . I f a p o i n t m u t a ti o n h a d o c c u r r e d
p r e c o c i o u s l y , t h is w o u l d i m p l y t h a t b o th M C H a n d c t - M S H w o u l d
h a v e h a d a P h e - A r g p a r t o f th e s e q u e n c e i n c o m m o n . T h i s w o u l d
b e v e r y i n t e re s t i n g b ecau se t h i s p a r t o f t h e seq u en ce (Ph e7 -A rg a ) i n
c t - M S H a n d ( T y r t l - A r g ] 2) i n M C H i s c o n s i d e r e d e x t r e m e l y
i mp o r t an t fo r t h e i r re sp ec t i v e ac t i v i t i e s (2 , 1 5 , 1 8 ) . Ce r t a i n l y , i t
w o u l d b e i n t e r es t i n g t o s e e i f c o n v e r s i o n o f T y r ] ~ t o P h e ~ i n M C H
w o u l d l ead t o an i n c rea se i n MSH -l i k e ac t i v i t y . In ad d i t i o n , t h i s
w o u l d b e i n t e r e s t i n g f r o m a n e v o l u t i o n a r y s t a n d p o i n t . W e c u r -ren t l y a re t e s t i n g t h i s h y p o t h es i s .
T h e f a c t th a t M C H h a s t h e a b i l it y t o e x h i b it M S H - l i k e a c t i v it y
l e a d s o n e t o c o n s i d e r a f u n c t i o n a l h o m o l o g y b e t w e e n t h e t w o
p ep t i d e s . In o t h e r w o rd s , t h e re may b e a t o p o g rap h i ca l s i mi l a r i t y
i n s t ru c t u re d e sp i t e a l a ck o f an y p r i mar y seq u e n ce h o m o l o g i e s .
O n e m a y p r o p o s e t h a t t he M S H - l i k e a c t i v i ty o f M C H c o u l d b e d u e
t o th e t r a n s d u ci n g c o m p o n e n t o f t h e M S H r e c e p t o r h a v i n g a
s i mi l a r t o p o l o g y , b u t a d i f fe ren t (b i n d i n g ) p o t en cy req u i remen t
t h a n t h e M C H r e c e p t o r ( o r v ic e v e r s a ). A l t e r n a t i v e l y , o n e c o u l d
p r o p o s e a c o m m o n r e c e p t o r t ha t m a y b e a c t i v a t e d i n d if f e r e n t w a y s
b y M C H a n d u - M S H . A l t h o u g h t h e s e c o n d a r g u m e n t a p p e a r s
u n l i k e l y (9 ) , n e i t h e r p ro p o sa l h a s b een u n eq u i v o ca l l y ru l ed o u t .
ACKNOWLEDGEMENTS
This work was supported by U.S. Public Health Service (AM 17420,
V.J.H.) and the National Science Foundation (DCB-8615706, M.E.H.),and by FAPESP (87/0851-4) and by CNPq (407196/87, 408683/88), Brazil(A.M .C.). The technical assistance of Mr. Michael Shea, Ms. RachelStover, M r. Sean Sullivan, and the clerical assistance of Ms, Dan ielleDuBois are gratefully acknowledged. The authors also wish to acknowl-edge the Midwest Center for Mass Spectrometry, a National ScienceFoundation Regional Instrumentation Facility (Grant No. CEH 8211164).Terry O. Matsunaga wishes to thank the National Institute on Drug Abuse
(NIDA) for a post-doctoral fellowship grant I F 05371-01.
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