EVOLUCIÓN JURISPRUDENCIAL DEL DERECHO DE ACCESO A LA

E x p erim en ts with the C D -M PR indicated that a ty ro sin e-b a se d endocytosis m otif is not the only functional internalization signal, and that in addition to the YRGV sequence, six residues, FPHLAF, also function as an endocytosis signal for this receptor (Johnson e t a l 1990). Both signals m ust be p resen t for m axim al rec ep to r in te r n a liz a tio n . H o w ev e r, m em b ran e p ro te in s w h ic h la c k a ty ro sin e -b a se d in te rn a liz a tio n signal also u n d e rg o e n d o c y to sis. These include the CD3y chain (Letoum eur and Klausner 1992), CD4 ( P e l c h e n - M a t t h e w s et al 1989; 1991; 1992; 1993; M arsh et al 1990), the Fc receptor (FcRII-B 2) (M iettinen et al 1989; 1992), and the a d ip o c y te /sk e le ta l m u scle glu co se tra n sp o rte r, G L U T 4 (Corvera et al 1994).

W hen the cy toplasm ic dom ain of CD3y was fused to the Tac antigen (IL-2 receptor a chain), some of the chimeric protein was delivered to the p lasm a m em brane, w h ilst the m ajo rity o f the protein was found in the lysosom es (L eto u rn eu r and K la u sn er

1992). The sequence which appeared to be m e d iatin g the delivery of this chimeric protein to lysosom es was D K Q T L L , of which the di-leucine was shown to be specifically required. This signal, now term ed a d i-leu cin e m otif, was also a ctiv e as an

en d o cy to sis signal, internalizing 90% of prebound iodinated anti- Tac antibody in 60 min (Letourneur and Klausner 1992).

Both CD4 and FcR II-B 2 are endocytosed through c o ated pits ( P e lc h e n -M a tth e w s et al 1991; 1993; Miettinen et al 1989; 1992), and both possess a di-leucine within their cytoplasm ic dom ains. The di-leucine in CD4 has been shown to be required fo r down- regulation (Shin et al 1991), and as this dow n-regulation appears to occur by endocytosis through coated pits (Section 3.1 P e l c h e n - M a tth e w s et al 1993), it is po ssib le th at the d i-le u cin e m ay function as an endocytosis signal.

l.S.d Entry Into The Endocytic Pathway From The Trans- Golgi Network.

In addition to sorting receptors and their ligands en d o cy to sed from the p lasm a m em brane, the endosom es are also resp o n sib le fo r so rtin g p r o te in s d e liv e re d d ire c tly fr o m th e s e c r e to r y pathway. The best documented pathway for the direct delivery of receptors from the secretory pathw ay to the en dosom es is that used by the CI-MPR. Both the CI-MPR and CD-MPR, are primarily responsible for the direct delivery of newly synthesized lysosom al hydrolases from the secretory pathway to the endocytic pathw ay w ith o u t appearing at the plasm a m em brane (D ahm s et al 1989; K ornfeld and M ellm an 1989; Ludwig et al 1991; Rijnboutt et al 1992). The C I-M P R is also resp o n sib le for the u p tak e and d e liv e ry to the en d o cy tic p athw ay of e x tra c e llu la r ly s o s o m a l h y d r o l a s e s .

In contrast to the lysosomal hydrolases, the delivery route o f the lysosom al m em brane glycoproteins (Igp's) from the TGN rem ains u n d er q u e stio n . In a sim ilar m an n e r to new ly s y n th e siz e d lysosom al hydrolases, transport o f rat l g p l2 0 from the T G N to lysosomes appears to occur via a direct intracellular route w ithout appearing at the plasm a m em brane (H arter and M ellm an 1992). H ow ever, newly synthesized lg p l2 0 was d etected at the plasm a m em brane when high levels of lg p l2 0 were expressed. T hese results indicated that direct intracellular delivery of l g p l2 0 from the T G N to the endocytic pathw ay was the m ajor route. In contrast to these observations, studies on the hum an ly so so m al m em brane glycoprotein, la m p -1, have in d ic ate d th at it is first

e n d o c y to s e d in to the end o cy tic p a th w a y and is d e liv e re d to ly s o s o m e s (W illia m s and F u k u d a 1990). T h is is a sim ila r m ec h an ism o f delivery to lysosom es as that used by lysosom al acid phosphatase (Braun et al 1989), and LEPlOO (M athew s et al 1 9 9 2 ). T h u s it a p p ea rs as th o u g h l y s o s o m a l m e m b r a n e g ly c o p ro te in s are d e liv ered to ly so so m e s e ith e r via the cell su rfa ce and entry into the endocytic p ath w ay , or th ro u g h the direct intracellular route similar to that taken by the CI-MPR.

l.S .e L y s o s o m a l T a r g e tin g S ig n a ls ,

As previously outlined above lysosom al m em brane glycoproteins (Igp's) can reach the lysosomes either by an intracellular route or via the plasm a membrane. The delivery of L A P and lamp-1 to lysosom es from the cell surface requires a tyrosine residue in the cytoplasm ic domain, that comprises p art of an endocytosis signal. M utation of the tyrosine to another residue such as phenylalanine or alanine, resulted in an accumulation of LAP and la m p -1 at the plasm a m em brane (Peters et al 1990; W illiams and Fukuda 1990), and m u ta tio n o f the c y to p lasm ic d o m ain ty ro sin e re s id u e at p o sitio n 8 in the rat l g p l2 0 to cy stein e, b lo ck ed d eliv ery o f l g p l2 0 to ly so so m e s and l e d to in c re a s e d l g p l2 0 p la s m a m em brane levels (Harter and M ellm an 1992). In addition to the ty ro sin e in the cytoplasm ic dom ain o f ra t l g p l2 0, an a d jacen t g ly cin e resid u e (position 7) also a p p ears to be re q u ire d for efficient lysosomal sorting. Similarly, m utation o f glycine 412 to alanine in LAP, leads to increased expression o f LAP at the cell surface (Lehm ann et al 1992). These results indicate that the GY residues in the cytoplasmic domains o f rat l g p l2 0 and LA P form p art of a lysosom al sorting signal, and that the tyrosine residue can also function in endocytosis. A num ber o f d ifferen t Igp's possess the GY m otif in their cytoplasmic dom ains, but it has yet to be d em o n strated that this m o tif fu n ctio n s as a ly so so m a l targeting signal for these other proteins.

Other lysosomal targeting signals which have been identified are the D K Q TLL and YQPL sequences in the CD3y chain of the TCR (L etourneur and Klausner 1992). Both the YQ PL and D K Q T L L signals were indiv id u ally su fficien t to in d u ce e n d o cy to sis and d e liv ery of the Tac antigen to ly so so m es. The targ etin g o f

C D -M P R s, and in C I-M P R this targ etin g is d e p en d e n t on the sequences LLH V and YSKV (Johnson and Kornfeld 1992), W hen these sequences were deleted from the cytoplasm ic dom ain o f CI- M PR , the targ etin g o f C athepsin D w as c o m p lete ly in h ib ited . How ever, deletion o f ju s t the LLHV sequence increased the levels o f C I-M PR reaching the plasm a m em brane, suggesting that the d i­ leucine m ight function in sorting C I-M PR from the T G N to the endocytic pathway. This di-leucine, although similar to th at in the C D 3 Y chain in terms o f intracellular sorting, does not appear to function in CI-MPR endocytosis (Lobel et al 1989).

T he re la tiv e im p o rta n c e o f the le u c in e -b a s e d sig n al in the targeting o f proteins to lysosom es has been suggested in studies with the type II lysosom al integral m em b ran e protein, L IM P II (V ega et al 1991). The C-term inal cytoplasm ic dom ain o f this protein con tain s a leu cin e -iso leu c in e d ip ep tid e at p o sitio n s 475 and 476, respectively, that is critical for the targeting of LIM P II to lysosom es (Sandoval et al 1994). T im e-course studies o f the d is trib u tio n o f a series o f p o in t m u ta n ts in d ic a te d th a t the dipeptide signals LI, LL, LV, LA or II could target L IM P II to lysosom es to different extents. In addition, N M R analysis o f an ico sap e p tid e which c o rresp o n d ed to the cy to p lasm ic d o m ain of L IM P II, rev ealed th at in solution this p ep tid e ad opted eith er r a n d o m c o il c o n fo r m a tio n s or t r a n s ie n t c o n f ig u r a tio n s . In particular, the segm ent L IR adopted a conform ation close to the values of an a helix, suggesting that the lysosomal targeting signal L I in L IM P II is w ithin a dom ain th at form s an e x te n d e d c o n fig u r a tio n .